Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

ABSTRACT

Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A  Neisseria meningitidis  using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/201,774, filed Aug. 11, 2005, now abandoned which application claimsbenefit of U.S. Provisional Application No. 60/600,862, filed Aug. 11,2004, both of which applications are incorporated by reference herein.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with funding from theNational Institutes of Health (Grant No. A140247) and the Department ofEnergy (Grant No. DE-FG02-93ER20097). Accordingly, the United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

The field of this invention is the area of molecular biology, inparticular as related to recombinant expression of an acetyltransferaseof Serogroup A Neisseria meningitidis, and immunogenic compositions,especially immunogenic compositions comprising fully acetylated capsuleof Neisseria meningitidis, Serogroup A.

Neisseria meningitidis is a leading worldwide cause of meningitis andrapidly fatal sepsis in otherwise health individuals (Apicella, M. A.(1995) in Principles and Practice of Infectious Diseases, eds. Mandell,G. L., Douglas, R. G., and Bennett, J. E., Churchill Livingstone, NewYork, pp. 1896-1909). In excess of 350,000 cases of meningococcaldisease were estimated to have occurred in 1995 (WHO Report (1996) WHO,Geneva, ISBN 92 4 1561823). The problem of meningococcal disease isemphasized by the recurrence of major epidemics due to serogroups A, B,and C N. meningitidis over the last 20 years, such as: the devastatingserogroup A outbreak in sub-Saharan Africa in 1996 (WHO (1996)Meningitis in Africa. The constant challenge of epidemics. WHO 21:15March); the recent dramatic increases in the incidence of serogroup Band C meningococcal disease in parts of North America (CDC (1995) MMWR44:121-134; Jackson, L. A. et al. (1995) JAMA 273:390-394; Wahlen, C. M.et al. (1995) JAMA 273:383-389); and the emergence in Europe andelsewhere of meningococci with decreased susceptibility to antibiotics(Campos, J. et al. (1992) J. Infect. Dis. 166:173-177).

Differences in capsular polysaccharide chemical structure determine themeningococcal serogroups (Liu, T. Y. et al. (1971) J. Biol. Chem.246:2849-58; Liu, T. Y. et al. (1971) J. Biol. Chem. 246:4703-12).Meningococci of serogroups B, C, Y, and W-135 express capsules composedentirely of polysialic acid or sialic acid linked to glucose orgalactose (Liu, T. Y. et al. (1971) J. Biol. Chem. 246:4703-12;Bhattacharjee, A. K. et al. (1976) Can. J. Biochem. 54:1-8), while thecapsule of group A N. meningitidis is composed of N-acetylmannosamine-1-phosphate (Liu, T. Y. et al. (1971) J. Biol. Chem.246:2849-58). The currently available capsular polysaccharide vaccinesfor serogroups A, C, Y, or W-135 N. meningitidis are effective forcontrol of meningococcal outbreaks in older children and adults.However, because of poor immunogenicity in young children andshort-lived immunity (Zollinger, W. D. and Moran, E. (1991) Trans. R.Soc. Trop. Med. Hyg. 85:37-43), these vaccines are not routinely usedfor long-term prevention of meningococcal disease.

In some epidemic settings, simultaneous or closely-linked meningococcaloutbreaks have occurred in the same population due to differentserogroups (Sacchi, C. T. et al. (1994) J. Clin. Microbiol.32:1783-1787; CDC (1995) MMWR 44:121-134; Krizova, P. and Musilek, M.(1994) Centr. Eur. J. Publ. Hlth 3:189-194). Further, Caugant et al.(Caugant, D. A. et al. (1986) Proc. Natl. Acad. Sci. USA 83:4927-4931;Caugant, D. A. et al. (1987) J. Bacteriol. 169:2781-2792) and othershave noted that meningococcal isolates of different serogroups may bemembers of the same enzyme type (ET)-5, ET-37 or ET-4 clonal complexes.

Neisseria meningitidis serogroup A is responsible for the massiveepidemics of meningococcal meningitis and septicemia that periodicallyaffect sub-Saharan Africa, China, South America and other parts of theworld. The serogroup A capsular polysaccharide (CPS) that confersserogroup specificity is composed of repeating units of (α1→6) linkedN-acetyl-D-mannosamine-1-phosphate that is O-acetylated (1). Althoughthere is evidence of other glycosidic linkages (2), the principallinkage between monomer ManNAc residues in this polysaccharide is the(α1→6) phosphodiester bond involving the hemiacetal group of carbon 1and the alcohol group of carbon 6 of the mannosamine residues. SerogroupA CPS is structurally distinct from other disease-causing meningococcalserogroups B, C, Y and W-135 which are composed of, or contain sialicacid (1, 3, 4).

There is a long felt need in the art for improved immunogeniccompositions useful for generating a protective immune response toNeisseria meningitidis, which is highly contagious and causes seriousillness.

SUMMARY OF THE INVENTION

The present invention provides recombinant DNA molecules which do notoccur in nature, recombinant host cells and methods of using theforegoing to recombinantly produce an O-acetyltransferase derived fromNeisseria meningitidis. This acetyltransferase transfers acetyl moietiesto capsular polysaccharides, especially those of Serogroup A N.meningitidis. The acetyltransferase of the present invention can bepurified using specific antibody in an immunoaffinity column, forexample, or an affinity tag can be engineered into the recombinantprotein by the use of appropriate tag (especially a polyhistidine or Histag) coding sequences fused in frame. Other oligopeptide “tags” whichcan be fused to a protein of interest by such techniques include,without limitation, strep-tag (Sigma-Genosys, The Woodlands, Tex.) whichdirects binding to streptavidin or its derivative streptactin(Sigma-Genosys); a glutathione-S-transferase gene fusion system whichdirects binding to glutathione coupled to a solid support (AmershamPharmacia Biotech, Uppsala, Sweden); a calmodulin-binding peptide fusionsystem which allows purification using a calmodulin resin (Stratagene,La Jolla, Calif.); a maltose binding protein fusion system allowingbinding to an amylose resin (New England Biolabs, Beverly, Mass.); andan oligo-histidine fusion peptide system which allows purification usinga Ni²⁺-NTA column (Qiagen, Valencia, Calif.).

The present invention further encompasses the acetylation (in vitro) ofSerogroup A capsular polysaccharides isolated from N. meningitidis usingacetyltransferase recombinantly produced using the recombinant hostcells of the present invention.

The present invention also provides for improved immunogeniccompositions comprising capsular polysaccharides of N. meningitidis,where the improvement comprises more complete acetylation of thecapsular polysaccharides than is currently possible in the absence ofthe enzymatic acetylation by using the acetyltransferase of the presentinvention, especially those from Serogroup A N. meningitidis, with theresult that a stronger immune response results. The immunogeniccompositions of the present invention can comprise a pharmaceuticallyacceptable carrier and optionally can further comprise at least oneimmunological adjuvant or cytokine. These immunogenic compositions areuseful as vaccines and as vaccine components. Desirably, the CPS is90-95% acetylated for eliciting a robust immune response

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genetic organization and location of the Neisseriameningitidis serogroup A capsule biosynthetic locus mynA-mynD(sacA-sacD), and the sites of polar (∇) and nonpolar (⋄) mutations inthese genes. ctrA is the first gene of the capsule transport operon andgalE encodes the UDP-glucose-4-epimerase.

FIG. 2A-2C provide ¹H NMR spectra of capsular polysaccharides (CPSs)purified from the wild type serogroup A meningococcal strain F8229 (FIG.2A) the mynC::aphA-3 mutant (FIG. 2B) and the mynC::aphA-3 mutantcomplemented with (mynC), with IPTG induction (FIG. 2C). Insets are theenlargements of the N/O—Ac methyl proton regions.

FIG. 3 demonstrates over-expression and purification of MynC ofserogroup A N. meningitidis. Lane 1, molecular weight marker; 2, celllysate before IPTG induction; 3, cell lysate after IPTG induction; 4,cell lysate on the nickel-nitrilotriacetic acid (Ni-NTA) column 5, flowthrough of the Ni-NTA column; 6, wash 1; 7, wash 2; 8, wash 3; 9,elution using 250 mM imidazole containing buffer. Arrow indicates the˜26 kDa His tagged MynC protein.

FIG. 4 is an autoradiogram showing the in vitro O-acetyltransferaseactivity of MynC. ¹⁴C-labeled acetyl coenzyme A and meningococcal CPSswere the substrates. Lanes (1) sample buffer alone, lanes 2-4 reactionsfrom various acceptor polymers plus 5 μg MynC (2) serogroup B CPS, (3)serogroup C CPS, (4) CPS of the serogroup A wild type strain F8229, (5)and (6) partially purified CPS of F8229/mynC::aphA-3 mutant at 5 and 10μg MynC respectively, (7) and (8) SEPHACRYL 200 gel filtration columnpurified CPS of F8229/mynC::aphA-3 mutant with 5 and 10 μg MynCrespectively, (9) column purified CPS of F8229/mynC::aphA-3 mutant withproteins eluted from a lysate of the E. coli strain carrying the pET20bvector without insert, and (10) column purified CPS of theF8229/mynC::aphA-3 mutant alone. Each reaction was performed with 50 μgof CPS and 10 μg of MynC, and analyzed as described in experimentalprocedures.

FIGS. 5A-5C characterize O-acetyltransferase activity of purified MynCas measured by ¹⁴C incorporation. FIG. 5A demonstratesconcentration-dependent incorporation of the ¹⁴C-labeled acetyl moietyby MynC into the mynC::aphA-3 CPS. FIG. 5B shows time kinetics of theincorporation of the ¹⁴C-labeled acetyl moiety into the mynC::aphA-3 CPSby MynC. FIG. 5C shows the pH optima of MynC activity in citrate (4.5 to6.5), phosphate (5.8 to 8) and borate (8.5 to 10.5) buffers.

FIG. 6A shows whole cell ELISA with mAb 14-1-A. 1, wild type parentF8229; 2, unencapsulated strain F8239; 3, mynC::aphA-3 nonpolar mutant;4, mynC::aphA-3 nonpolar mutant complemented with pGS205 (mynC) in theabsence of IPTG induction; and 5 mynC::aphA-3 nonpolar mutantcomplemented with pGS205 (mynC) in the presence of IPTG induction. FIG.6B illustrates Western blot analysis with whole cell lysatesdemonstrating the His-tagged MynC in the presence (+) or absence (−) ofIPTG. Lanes 1, M.W. marker (32.3 KDa); 2, wild type strain F8229; 3 and4, overexpressed wild type strain NmAwtc1; 5 and 6, complementednonpolar mutant NmAnpc1.

FIG. 7A shows the cellular localization of MynC. Western blot analysisof sub-cellular fractions of mynC complemented strain NmAnpc1 using(His)₅ mAb. The loadings of individual fractions were standardized basedon a set amount of cells obtained from 500 ml culture. FIG. 7Bdemonstrates peripheral and strong membrane association of MynC. Totalmembrane obtained from NmAnpc1 cells was extracted with buffer alone, 1MNaCl, 6M urea or buffer with 1% TX-100 as described in the Materials andMethods. After centrifugation, soluble fraction (S) were concentrated byprecipitation with trichloroacetic acid, pellets (P) were resuspendeddirectly in sample buffer. Fractions were subjected to 10% SDS-PAGE gelsand analyzed by western blots using (His)₅-specific mAb.

FIGS. 8A and 8B show the coding and amino acid sequences for the N.meningitidis mynC, respectively. See also SEQ ID NO:1 and SEQ ID NO:2,respectively.

FIG. 9 shows the results of a normal human serum (10% v/v) bactericidalactivity assay with the OAc+/CAP+ N. meningitidis wild-type parent F8229(lane 1), the serogroup A CAP− strain F8239 (lane 2), the CAP− mutantsof strain F8229 (mynA, lane 3; mynB, lane 4) and the OAc−/CAP+mynC::aphA3 (lane 5) mutant of F8229. Percentage of meningococcalsurvival in the presence of normal human serum (black bars) and in thepresence of heat inactivated (56° C., 30 min) serum (gray bars) isshown.

FIGS. 10A and 10B show the results of competitive inhibition ELISAsperformed using purified CPS of the serogroup A N. meningitidisOAc+/CAP+ wild-type parent F8229 (FIG. 11A) and the OAc−/CAP+mynC::aphA3 mutant (FIG. 11B), and sera obtained from six differentindividuals (numbered in side legend) previously vaccinated with alicensed vaccine containing the serogroup A polysaccharide.

FIGS. 11A-11B provide a comparison of ¹H NMR spectra of the anomeric andthe ring proton regions of serogroup A N. meningitidis wild type parentstrain F8229 using the isolated CPS at 500 MHz (FIG. 11A) and wholecells by HR-MAS at 600 MHz (FIG. 11B).

FIGS. 12A-12C provide comparisons of whole cell HR-MAS ¹H NMR patternsin the anomeric and ring proton regions of serogroup A N. meningitidiswild type parent F8229 (FIG. 12A), capsule O-acetylation negative mutantstrain NMA001 (FIG. 12B) and capsule negative serogroup A strain F8239(FIG. 12C). FIG. 12D-12F provide a comparison of whole cell HR-MAS ¹HNMR patterns in the O—Ac, N—Ac methyl proton region of serogroup A N.meningitidis wild type parent F8229 (FIG. 12D), capsule O-acetylationnegative mutant strain NMA001 (FIG. 12E) and capsule negative serogroupA strain F8239 (FIG. 12F).

DETAILED DESCRIPTION OF THE INVENTION

The abbreviations used herein are CPS, capsular polysaccharide; O—AcCPS, O-acetylated capsular polysaccharide; PCR, polymerase chainreaction; GC-MS, gas-liquid chromatography-mass spectrometry; COSY,¹H-¹H correlation spectroscopy; TOCSY, total correlation spectroscopy;High Resolution Magic Angle Spinning NMR Spectroscopy, HR-MAS NMR; mAb,monoclonal antibody; ELISA: enzyme linked immunosorbant assay; SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DOC-PAGE,deoxycholate-polyacrylamide gel electrophoresis; ManNAc, N-acetylmannosamine.

Capsular polysaccharide is the critical virulence determinant in N.meningitidis and Four (A, C, Y, and W-135) of the five clinicallyimportant meningococcal disease causing serogroups express O-acetylatedcapsules (1, 3, 30, 31). We describe herein the identification of theserogroup A CPS biosynthetic gene mynC and its gene product MynC. MynCis required for serogroup A meningococcal capsular O-acetylation; it isthe O-3 and O-4 N-acetyl mannosamine acetyltransferase. MynC representsa new class of O-acetyltransferase with no homology with knownO-acetyltransferases or the proposed sialic acid capsular serogroup C,Y, and W-135 meningococcal O-acetyltransferases OatC or OatWY reportedrecently (5). MynC is an inner membrane-associated protein with notransmembrane domains. It seems to be a peripheral protein having tightassociation with the inner membrane and could be disrupted only bystringent 6 M urea wash and not by a more mild 1 M NaCl wash. Theinability of TX-100 condition to extract the MynC off the membrane,confirms that this protein is not an integral membrane protein as alsoindicated by transmembrane domain search. The strong association of MynCwith the membrane suggests that this protein could be a component ofmulti-protein complex engaged in capsule biosynthesis.

O-acetylation of bacterial surface polysaccharides such as capsularpolysaccharides, exopolysaccharides, peptidoglycans andlipooligosaccharides is common in pathogens and in symbionts,O-acetylation has immunogenic and functional importance. N.meningitidis, K1 E. coli, S. pneumoniae, Salmonella enterica,Staphylococcus aureus and Pseudomonas aeruginosa can expressO-acetylated CPS (31,32). In S. enterica serovar typhi (7) and in E.coli K1 (6), the loss of O-acetylation from CPS results in loss ofimmunogenicity, whereas for meningococcal serogroup C (30) andpneumococcal type 9V (33) capsules, O-acetylation is not required forthe induction of protective antibodies. In the extracellularpolysaccharide alginate polymer, produced by isolates of P. aeruginosafrom patients with cystic fibrosis, D-mannuronic acid is O-acetylated atO-2 and at O-3 by three genes algI, algJ, and algF (34). AlginateO-acetylation had been shown to contribute to biofilm architecture,microcolony formation (35) and resistance to opsonic phagocytosis (36).O-acetylation is also important for rhizobium-legume symbiosis. Therhizobial Nod factors may be O-acetylated at distinct sites to definethe host specificity and the formation of the pre-infection thread andthe root nodule (37-39). In Proteus mirabilis, N. gonorrhoeae and N.meningitidis (40), C-6 hydroxyl of N-acetyl muramyl residues inpeptidoglycans are O-acetylated to confer both intrinsic and completeresistance to lysozyme hydrolysis. These peptidoglycan motifs arepathogen-associated molecular patterns recognized by the innate immunesystem (41,42).

A number of acetyltransferases that transfer an acetyl group fromacetyl-CoA to O-acetylate dissimilar substrates have been identified inprokaryotic and eukaryotic systems but these proteins share limitedsequence homology. Two families of proteins that O-acetylate exportedcarbohydrate moieties have been reported. The NodL-LacA-CysE family(43-47) that include the lipochitin acetyltransferase (NodL) ofRhizobium leguminosarum, galactoside acetyltransferases (GAT) such asLacA, the cysteine biosynthetic enzyme (CysE), also known as the serineacetyltransferase of E. coli, are cytoplasmic proteins that use acetylcoenzyme A as the acetyl donor. Interestingly, the proposed sialic acidO-acetyltransferases of meningococcal serogroups W-135 and Y (OatWY) butnot of serogroup C (OatC) show sequence homology to the NodL-LacA-CysEfamily. The second family comprises integral membrane proteins. Membersof this family include the O-acetyltransferases that O-acetylatemacrolide antibiotics (Streptomyces spp.) (48), LPS O-antigen(Legionella pneumophila Lag-1, (49) Salmonella typhimurium OafA (50),Shigella flexneri bacteriophage SF6 OAc (51) and Nod factors (Rhizobiumleguminosarum NodX (52). However, the putative capsuleO-acetyltransferases (50) of Streptococcus pneumoniae serotype 9V,Cps9vM and Cps9vO the S. aureus serotype 5 O-acetyltransferase (53) andalginate O-acetylation proteins AlgI, AlgJ and AlgF of P. aeruginosashare no homology with the above mentioned families ofO-acetyltransferases. Similarly, MynC represents a novel subclass ofacetyltransferases.

The enzymatic activity for capsular polysialic acid O-acetylation fromK1 E. coli was reported by Higa and Varki (54), but the respective geneand the protein have not been identified. MynC does show sequencehomology with several proteins (Table 2), including the acetyl esterase(acetyl xylosidase) that degrades xylan from the thermophile,Caldicellulosiruptor saccharolyticus. These proteins share with MynC asemi-conserved motif GSSKGG (SEQ ID NO:12) in the N-terminal region.Typically, serine esterases contain a conserved GSSSG (SEQ ID NO:13)motif (assumed to be the catalytic N-terminal domain), where the middleS residue is the active site nucleophile (55). MynC also has homology(25% identity and 46% homology) with capsule biosynthesis enzyme Cap8I(464 aa) of S. aureus subsp. aureus MW2 (27) and to a hypotheticalesterase/lipase/thioesterase family protein (265 aa) of Arabidopsisthaliana. The S. aureus serotype 8 capsule has O-acetylation in themannuronic acid component of the capsule.

A BLAST search performed with the deduced MynC (247 aa) amino acidsequence (SEQ ID NO:2), identified five proteins in the Gen Bank with25% sequence identity (Table 2). Among these were EpsK of Lactococcuslactis subsp. cremoris, acetyl esterase/xylosidase (EC 3.1.1.6, 266 aa)XynC of Caldicellulosiruptor saccharolyticus (26), and a capsularpolysaccharide synthesis protein, Cap8I (464 aa), from Staphylococcusaureus subsp. aureus MW2 (27). Interestingly, these five proteins sharedwith MynC a semi-conserved motif (GSSKGG) SEQ ID NO:12 of mostlyhydrophobic small amino acids in the N-terminal region. Repeated searchand pairwise comparison of known O-acetyltransferases from prokaryotesand eukaryotes revealed no significant homology with MynC.

A motif scan search of the MynC sequence at ISREC (Swiss Institute forExperimental Cancer Research) and SIB (Swiss Institute forBioinformatics) sites revealed no matches. Search results using theSIB-PROSITE database of protein families and domains showed nosimilarity. Using a Markov model for transmembrane domain prediction,TMHMM (Centre for Biological Sequence Analysis, Technical University ofDenmark, Lyngby, Denmark) MynC has no transmembrane domains. EMBL-EBI(European Bioinformatics Institute) InterProScan predicted MynC as amember of alpha/beta-hydrolases super-family that includesacetylcholinesterases, carboxylesterases, mycobacterial antigens, andacetylesterases.

Growth of the mynC nonpolar mutant was not different in GC medium whencompared with the wild type parent. However, when the pellets from oneliter cultures of similar growth (OD₆₀₀ of 1.0) were compared for CPSyields, the mynC mutant consistently yielded 25-30% less CPS compared tothe wild type parent, probably due to some polarity of the insertionmutant or due to a decrease of transcript stability. Capsularpolysaccharides from the wild type strain F8229 and the nonpolar mynCmutant NMA001 were prepared, purified and subjected to compositional andstructural analysis. The GC-MS analysis of the alditol acetatederivatives, after removal of the phosphate groups by HF treatment,revealed ManNAc as the sole component of capsular polysaccharidesisolated from both the wild type strains and the mynC mutant.

In order to investigate the extent of O-acetylation and the location ofthe O-acetyl groups, the CPSs were subjected to 1-D and 2-D ¹H NMRspectroscopic analyses. Assignments of the various protons could be madefrom the COSY and TOCSY NMR analyses. The wild type CPS 3-O—Ac protonassignments (Table 3) were compared to published values (28,29) and werehighly consistent with these values. However, the mynC mutant CPSspectrum was quite distinct.

In the wild type CPS ¹H NMR spectrum shown in FIG. 2A, the H-3 proton ofManNAc was observed at 5.20 ppm when the moiety had acetylation at O-3due to the de-shielding effect of the acetyl group. The absence of thispeak in the spectrum of the mutant CPS (FIG. 2B) indicated the lack ofacetylation at O-3 on the ManNAc residue. The H-2 resonance at 4.61 ppmwas observed in the wild type CPS indicating 3-0 acetylation, whereas inthe mynC mutant spectrum this peak was missing (comparing FIGS. 2A-2B).In the region between 2.05 to 2.10 ppm where N- and O acetyl methylprotons were observed (inset, FIG. 2A, and Table 3) three peaks wereidentified in the wild type CPS spectrum. Two of these peakscorresponded to O-acetyl methyl protons, while the other was due toN-acetyl methyl protons. However, in the spectra (inset, FIG. 2B andTable 3) of the mynC mutant CPS only one peak corresponding to theN-acetyl methyl proton resonance at 2.08 ppm was observed, suggestingthe absence of O-acetylation. These differences in 1-D NMR spectraindicated the absence of O-acetylation in the mynC mutant CPS.

The relative percentages of the CPS populations (Table 4) from the wildtype parent and mynC mutant were calculated using integration values ofthe H2 resonance (28,29). Integration of the ManNAc H2 resonances forthe various CPSs revealed that wild type CPS consisted of 3-O—Ac (4.59ppm), 4-O—Ac (4.54 ppm when adjacent to 3-O—Ac-ManNAc and 4.50 ppm whenadjacent to non-O-acetylated ManNAc) and Non-O—Ac (4.45 ppm) forms inthe ratio of 4:2.7:3:3, and this value was found to be consistent amongdifferent batch preparations. CPS of the mynC mutant showed a 100%non-O—Ac form (peak at 4.45 ppm). In conclusion, absence of both 3 and 4O-acetylation in mutant CPS suggested that MynC was responsible for theO-acetylation at both positions.

To further confirm the NMR data, a colorimetric estimation (25) ofO-acetylation of triplicate samples of 400 and 1000 μg amounts ofpurified CPS from the wild type parent and mynC mutant was performed.The wild type CPS showed significant O-acetylation (at 500 nm OD±S.D of0.2138±0.015 and 0.4896±0.003, respectively) whereas the CPS of the mynCmutant yielded minimal absorbance (at 500 nm OD±S.D of 0.0553±0.014 and0.1400±0.028 respectively) likely due to N-acetylation.

In further studies of Serogroup A capsular polysaccharides, N.meningitidis cells were grown, and HR-MAS NMR analysis was performedfollowing the methods described previously (68). Briefly, bacteria grownovernight on GC-agar plates (˜10¹⁰ cells) were harvested and killed in 1ml of 10 mM potassium-phosphate buffer (pH 7.4) in D₂O containing 10%sodium azide (w/v). The suspension was incubated for 1 h at roomtemperature. The bacteria were pelleted by centrifugation (9700×g for 2min) and washed once with 10 mM potassium phosphate buffer in D₂O. Thepellet was mixed with 20 μl of D₂O containing 0.75% (w/v) TSP(3-(trimethylsilyl)-propionic acid-D₄, sodium salt) as an internalstandard (0 ppm) prior to being loaded into a 40 μL nano NMR probe(Varian, Palo Alto, USA). HR-MAS experiments were performed using aVarian Inova 600-MHz spectrometer. Spectra were spun at 3 kHz andrecorded at ambient temperature (21° C.). The experiments were performedwith suppression of the HOD signal at 4.8 ppm by presaturation. Protonspectra of bacterial cells were acquired with theCarr-Purcell-Meiboom-Gill (CPMG) pulse sequence(90-(τ-180-τ)_(η)-acquisition) to remove broad lines arising from lipidsand solid-like-material. The total duration of the CPMG pulse (n*2τ) was10 ms with τ set to (1/MAS spin rate). Typically spectra were acquiredeach with 400 acquisitions in approximately 15 min. with a recycle delayof 2.5 sec.

Purified meningococcal CPSs have been extensively investigated using ¹Hand ¹³C NMR spectroscopy (3, 61, 28) and O-acetylation patterns of CPSshave been validated using NMR techniques (29) for meningococcalpolysaccharide containing vaccines. We have described purified serogroupA CPS by ¹H NMR to identify the serogroup A O-acetyltransferase encodinggene mynC. When the O-3, O-4, OAc+ serogroup A wild type F8229meningococci were subjected to HR-MAS analysis (FIG. 12A), reproducibleserogroup A CPS derived proton resonances were noted. The respective CPSderived HR-MAS proton signals were easily correlated with the ¹H NMRsignals obtained from purified CPS (FIG. 12B) with identical chemicalshifts. The characteristic anomeric peaks corresponding to 3-O-acetylManNAc H1 and ManNAc H1 were observed at 5.46 and 5.44 ppm, respectivelyand the 3-O-acetyl ManNAc H2 and ManNAc H2 observed at 4.61 ppm and 4.4ppm, respectively. Wild type 3-O-acetylated ManNAc H3 signal wasobserved at 5.20 ppm and O-acetyl methyl protons were observed at 2.10and at 2.06 ppm (see FIGS. 12D-12F). To further validate these data, thewild type stain, a capsule O-acetylation deficient mutant of this strainand a capsule defective stain were studied by HR-MAS (FIG. 12A-12C).When compared to the wild type parent (FIG. 12A), the mynC mutantmeningococci gave a profile (FIG. 12B) lacking the peaks at 5.20 ppm,4.59 ppm, 2.10 ppm and 2.06 ppm typical of a non-O-acetylated serogroupA CPS. A comparison of an enlarged high field region, 2-2.18 ppm (FIG.12D-12F), confirmed the lack of OAc methyl proton signals in the mynCmutant spectrum (FIG. 12B) that showed a single N-acetyl methyl protonresonance at 2.08 ppm. The capsule-negative strain F8239 showed noresonance characteristic of CPS indicating the lack of capsule on thesurface (e.g., FIGS. 12C and 12F).

The degree of 3-O acetylation was estimated from peak integrals obtainedusing the standard Varian software. The relative amount of the 3-O—Acform of the CPS was calculated from integrals of the H-1 resonances at5.46 ppm (3-O—Ac ManNAc) and 5.41 ppm (ManNAc) and found to be 1.6:1(i.e. 57+/−3%). Additionally, comparison of the 3-O—Ac ManNAc H-3integral with that of the combined anomeric region gave 50+/−3% of the3-O—Ac form. These results agree well with the 50% 3-0 acetylationdescribed herein. See also Gudlavalleti et al. (69). The H-2 resonanceof the ManNAc residue was shown previously (28) to be sensitive to notonly 3-0 acetylation but also to acetylation at O-4 in the purifiedcapsular polysaccharide. Peaks at 4.59 ppm, 4.54 ppm and 4.50 ppm in theHR-MAS spectrum of whole cells are consistent with those reported forpurified CPS H2 of 3-O-acetylated ManNAc, 4-O—Ac-ManNAc adjacent to a3-O-acetylated ManNAc residue, and 4-O—Ac-ManNAc adjacent to anon-acetylated ManNAc residue, respectively. Although the peakintegration was less precise, the degree of 4-O-acetylation wasestimated to be half of the level of 3-O acetylation (i.e.,approximately 25% of the CPS). This result is in agreement with thelevel of 27% acetylation at the 4-O position of ManNAc in purifiedserogroup A CPS determined in an independent experiment. These studiesindicate that HR-MAS NMR technique can be applied to directly determineand quantitate the structures of CPS that are surface expressed.

To confirm the acetyltransferase activity in an in vitro assay, MynC wasHis-tagged at its C-terminus, over-expressed in E. coli and purified innative conditions using Ni-NTA affinity chromatography (FIG. 3). Thecolumn was washed with buffer containing 10, 20 and 40 mM imidazolerespectively. A 40 mM imidazole wash was required to remove highmolecular weight contaminating bands (lane 8, FIG. 3). Elution of MynCwith 250 mM imidazole containing buffer yielded a purified protein (lane9, FIG. 3).

Purified MynC was used in in vitro assays containing the serogroup Awild type or mynC mutant CPS as the substrate and(acetyl-1-¹⁴C)-coenzyme A as the acetyl donor. Autoradiography of theCPSs (FIG. 4) revealed that MynC transferred the ¹⁴C labeled acetylgroup from acetyl CoA to the non-acetylated CPS of the mynC mutant(lanes 5-8, FIG. 4). Interestingly, MynC was also capable of furtherO-acetylating the wild type CPS (lane 4, FIG. 4). MynC recognized theserogroup A CPS but not serogroup B or serogroup C CPSs (lanes 2 and 3,FIG. 4). Finally, the acetyltransferase activity was not due to minorcontaminating E. coli proteins left after purification, as the lysate ofthe vector construct alone did not exhibit activity (lanes 9 and 10,FIG. 4). MynC activity was concentration-dependent (FIG. 5A) when theamount of CPS substrate and the acetyl donor were constant. The decreasein the estimated activity over 2-3 h time point could be due to thepossible degradation of the CPS polymer at the reaction condition thathad been removed in 80% ethanol washes. The enzyme seems to be inactivein the extreme pH conditions of less than 5 and greater than 10. Theoptimal pH for the MynC activity was 5.8 to 7.0 (FIG. 5C). The Mg⁺² ionspresent in the in vitro O-acetyltransferase reaction buffer may not beessential for the enzyme activity, as revealed by the assay usingcitrate, phosphate and borate buffers without these ions, for optimal pHmeasurements.

An intact copy of mynC under the control of a lac promoter wasconstructed and sub-cloned into the meningococcal shuttle vector,pYT250, as described in Example 5. The plasmid was transformed into themynC mutant and the wild type strain to generate strains NmAnpc1 andNmAwtc1, respectively. The wild type strain and the wild type strainover-expressing MynC (NmAwtc1), the mynC mutant, and the complementedstrain (NmAnpc1) were grown on GC agar plates with or without IPTG andanalyzed by colony immunoblots and ELISAs (FIG. 6A) using the serogroupA capsule-specific monoclonal antibody 14-1-A. The unencapsulated strainF8239, a serogroup A strain which contains point mutations and deletionsin mynA (11) was used as a negative control. The wild type and NmAwtc1meningococci were strongly recognized by mAb 14-1-A, whereas theuncomplemented mynC nonpolar mutant and the capsule negative controlstrain F8239 did not react with this antibody. The complemented nonpolarmynC mutant strain NmAnpc1 reacted strongly, and the intensity wasincreased with IPTG induction. These data indicated that a O-acetylgroup was a component of the epitope specificity of the monoclonalantibody 14-1-A. The CPS isolated from complemented strain NmAnpc1 wassubjected to ¹H NMR analyses which revealed (FIG. 2C) the restoration ofO-acetylation in the polymer. When compared by the relative integrationvalues of H2 resonances of 3-O—Ac and non-O—Ac forms, the level ofO-acetylation in the complemented strain, even with IPTG, was less thanwild type levels, although the relative ratio (5:1:2) of the acetylatedspecies O-3: O-4: O-3 and 4 was similar to the wild type ratio (4:1:1.7)(Table 4). The mynC mutant CPS O-acetylated in vitro by MynC wasrecognized by the monoclonal antibody 14-1-A, confirming the importanceof O-acetylation in defining the epitope recognized by the antibody.

In summary, quantitative ELISA, ¹H NMR and colorimetric assays on theCPS from complemented strain NmAnpc1 revealed that O-acetylation wasrestored by genetic complementation. Western blot analysis (FIG. 6B) onthe whole cell lysates of the MynC over-expressed wild type strain(NmAwtc1), and the complemented mynC mutant (NmAnpc1), using the antipenta-His mAb, was also performed. His-tagged MynC (lanes 4 and 6, FIG.6B) was visualized in the complemented meningococci under IPTG and thisresult was correlated with the restoration of O-acetylation in the NmAmutant CPS.

The MynC-complemented strain NmAnpc1 was used to assess the cellularlocation of MynC in serogroup A meningococci. Western blot analysis ofthe sub-cellular fractions loaded on the basis of a set amount ofstarting cells, using (His)₅ mAb revealed (FIG. 7A) that MynC was innermembrane-associated. The total membrane and inner membrane componentsgave strong reactivity, whereas the cytosolic fraction showed weakreaction and outer membrane showed no reaction. To explore thepossibility MynC that did not possess any transmembrane domains to be aperipheral protein, various extraction procedures were performed.Treatment of membranes with 6M urea partially stripped off the protein,whereas the other conditions with 1M NaCl or with 1% Tx-100 did notextract the MynC (FIG. 7B) from the total membranes.

Cell surface hydrophobicity, a marker of capsular expression, wasmeasured by hydrophobic interaction column chromatography (23).Approximately 4% of the wild type serogroup A strain F8229 and about 5%of the mynC mutant were retained on the hydrophobic column, indicatingoverall low cell surface hydrophobicity of both the wild type and themutant. In contrast, the unencapsulated variant F8239 (>60%) and mynAand mynB mutants (>90%) were retained on the column, indicating highcell surface hydrophobicity. A bactericidal assay using 10% normal humansera was used to assess expression of a functional capsule. Both thewild type parent and the mynC nonpolar mutant were protected fromkilling. In contrast, the unencapsulated strain F8239 and the mutants ofmynA, mynB and mynD were completely killed under these conditions.

O-acetylation is critical for serogroup A N. meningitidis CPSimmunogenicity and antibody formation (8). The major protective epitoperecognized by antibodies induced following vaccination with serogroup Apolysaccharide requires O-acetylation. Berry et al. (8) found thatbactericidal anti-serogroup A antibodies in the sera of serogroup Apolysaccharide vaccinated individuals were specific for O—Ac CPS. Theimportance of O—Ac in serogroup A capsule immunogenicity was confirmedusing O—Ac and non-O—Ac PS and PS-protein conjugates in immunogenicitystudies with mice. Interestingly, the serogroup A capsule with orwithout O-acetylation protected the meningococcus against killing by alow concentration (10%) of normal human serum, e.g. antibody-independentcomplement mediated killing. O-acetylation may also have a role in theinitial stages of colonization and infection by serogroup A N.meningitidis. In studies of meningococcal colonization in a mouse model(56), an OAc− mynC mutant showed significantly lower ability toestablish colonization compared to the wild type OAc+ strain.

MynC is specific for meningococcal serogroup A {(α1→6) linkedN-acetyl-D-mannosamine-1-phosphate} CPS. MynC did not acetylate thesialic acid CPSs of either serogroup B or serogroup C N. meningitidis.The in vitro O-acetylation studies indicate that MynC recognized nonO-acetylated or the partly O-acetylated CPS assembled polymer as asubstrate. Therefore O-acetylation appears to be a near final step ofdecorating the serogroup A capsule polymer. The cell surfacehydrophobicity data and the resistance to killing by normal human seraof the mynC mutant and the wild type parent indicate that the OAc−capsular polymer is surface expressed and functional. Thus, serogroup Acapsule expression, transport or prevention of killing by normal humansera does not require O-acetylation.

In summary, MynC is the capsular polysaccharide O-3 and O-4acetyltransferase of serogroup A of N. meningitidis. This approximately25 kDa inner membrane associated enzyme utilizes acetyl CoA for itsactivity and belongs to a new subclass of O-acetyltransferases. Study ofthe OAc deficient mutant confirmed the importance of O-acetylation inserogroup A polysaccharide immunogenicity, but O-acetylation was notrequired for capsular expression or to protect the meningococcus fromkilling by normal human sera. O-acetylation by MynC may be important forvaccine development against serogroup A N. meningitidis. The ability toachieve O-acetylation of serogroup A polysaccharides used for new andexisting meningococcal conjugate and polysaccharide vaccines may beenhanced by this enzyme.

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) iscomposed of a homopolymer of O-acetylated, (α1→6) linkedN-acetyl-D-mannosamine (ManNAc)-1-phosphate that is distinct from thecapsule structures of the other meningococcal disease causing serogroupsB, C, Y and W-135. The serogroup A capsule biosynthetic genetic cassetteconsists of four ORFs, mynA-D (sacA-D) that are specific to serogroup A,but the function of these genes has not been well characterized. Wefound that mynC encoded an acetyltransferase that was responsible forthe O-acetylation of the CPS of serogroup A. The wild type CPS asrevealed by ¹H NMR had 60 to 70% O-acetylated ManNAc residues thatcontained acetyl groups at 0-3, with some species acetylated at O-4 andO-3 and O-4. A nonpolar mynC mutant, generated by introducing an aphA-3kanamycin resistance cassette, produced CPS with no O-acetylation. Aserogroup A capsule-specific monoclonal antibody was shown to recognizethe wild type O-acetylated CPS but not the CPS of the mynC mutant, whichlacked O-acetylation. MynC was C-terminally His-tagged and overexpressedin E. coli to obtain the predicted ˜26 kDa protein. Theacetyltransferase activity of purified MynC was demonstrated in vitrousing ¹⁴C labeled acetyl CoA. MynC, O-acetylated the OAc-CPS of the mynCmutant, and further acetylated the wild type CPS of serogroup A but notthe CPS of serogroup B or serogroup C meningococci. Geneticcomplementation of the mynC mutant confirmed the function of MynC as theserogroup A CPS O-3 and O-4 acetyltransferase. MynC is an innermembrane-associated protein of a new subclass of O-acetyltransferasesand utilizes acetyl CoA to decorate the D-mannosamine capsule ofserogroup A N. meningitidis.

Meningococcal serogroups C, Y, W-135 and H also express O-acetylatedcapsules. Interestingly, the serogroup B CPS is not O-acetylated. Thegenes indispensable for encoding the putative capsular polysaccharideO-acetyltransferases (OatC, OatWY) responsible for the O-acetylation ofmeningococcal serogroups C, W-135 and Y, respectively, have beenrecently identified (5). Other pathogens such as pneumococcal serotype9V, Salmonella enterica serovar typhi Vi, Staphylococcus aureusserotypes 5, 8 and E. coli K1(6) express O-acetylated capsules. Thebiological importance of O-acetylation of CPS appears species orsubspecies dependent but in some pathogens O-acetylation of capsule isinvolved in immune recognition (6,7). For serogroup A CPS there is adramatic reduction in immunogenicity of the polysaccharide observed withremoval of the O-acetyl groups by chemical treatment (8).

The general genetic organization of capsular polysaccharide genes of N.meningitidis is similar to other bacterial systems such as Haemophilusinfluenzae, E. coli K1, etc. that are classified (9,10) as group IIcapsules. It is composed of unique biosynthesis gene cassette flanked byconserved genes involved in translocation of the CPS. The geneticcassette responsible for the biosynthesis of the serogroup A capsule iscomprised of a ˜5 kb nucleotide sequence located (FIG. 1) between ctrA,the outer membrane capsule transporter, and galE, theUDP-glucose-4-epimerase (11). Four open reading frames (ORFs 1 to 4designated as myn A-D or sacA-D) are co-transcribed as an operon (11)and are not found in the genomes of other meningococcal serogroups or inNeisseria gonorrhoeae. Separated from ctrA by a 218-bp intergenicregion, mynA is predicted to encode a 372-amino acid protein that hashomology with the E. coli UDP-N-acetyl-D-glucosamine 2-epimerase, MynBis hypothesized to be the capsular polymerase, linking individualUDP-ManNAc monomers together and MynD was predicted to be involvedeither in CPS transport assembly or in cross-linking of the capsule tothe meningococcal cell surface (11). See also U.S. Pat. No. 6,403,306.In the present study we demonstrate that mynC (744-bp) encodes anO-acetyltransferase (247 aa) that transfers acetyl groups to the ManNAcresidues of the serogroup A CPS.

Serogroup A Neisseria meningitidis is a major cause of endemicmeningococcal disease as well as epidemics and pandemics ofmeningococcal meningitis and meningococcemia in many developing parts ofthe world. Capsular polysaccharide (CPS) of serogroup A N. meningitidisis composed of O-3 or O-4 acetylated a (1→6) linked phospho-ManNAcpolymers (1) and is distinct from the chemical structures of the othermeningococcal capsular polysaccharides. In serogroup A meningococcalpolysaccharide vaccines, O-acetylation of the serogroup A CPS isbelieved to be important for immunogenicity and protection (8). Otherroles of CPS O-acetylation in serogroup A meningococcal pathogenesishave not been defined. This applications discloses the serogroup A CPSO-acetyltransferase gene; and the genes involved in meningococcal sialicacid capsule O-acetylation have also been identified (5). Further,O-acetylation in serogroup A and the other meningococcal serogroups' CPSpatterns have been extensively elucidated and investigated by ¹³C NMRand ¹H NMR experiments (3, 61, 28).

The N. meningitidis serogroup A CPS biosynthesis genetic cassette iscomprised of a ˜4.7 kb (11) region containing four ORFs—mynA, mynB, mynCand mynD also known as sacA-D. MynC is responsible for the O-3 and O-4acetylation of ManNAc CPS. A nonpolar mutation in mynC, generated byinsertion of the aphA-3 kanamycin resistance cassette, yielded a CPSdevoid of O-acetylation. Colony immunoblots, cell surface hydrophobicitystudies and capsule precipitation procedures revealed that the nonpolarmynC mutant (mynC::aphA-3) surface expressed similar amounts of capsularpolysaccharide to the wild-type parent. In this study, the serogroup Aencapsulated wild-type parent F8229 (11), an isogenic OAc− nonpolarencapsulated mutant mynC::aphA3 and the unencapsulated mynA or mynBmutants of this strain and the serogroup A capsule deficient strainF8239 were used.

The role of O-acetylation and CPS in the ability of serogroup Ameningococci to colonize the nasopharynx of outbred adult Swiss Webstermice was tested. This model has previously been used to define a role ofserogroup B capsule in meningococcal colonization. Mice (5/group) wereinoculated with 10⁷ CFU of meningococci intra-nasally and were followedfor five days with nasopharyngeal washes and cultures of these washes.The wild-type parent (F8229 CAP+/OAc+) effectively colonized 75% ofmice, whereas the mynC CAP+/OAc− mutant initially colonized 50%. By day2, 15% of mice remained colonized with the mynC CAP+/OAc− mutant,whereas with the CAP+/OAc+ wild-type parent, 60% of mice remainedcolonized. By day 3, colonization of all mice inoculated with the mynCCAP+/OAc− mutant was lost. In contrast, 22-30% of mice inoculated withthe wild-type parent remained colonized through the five days ofobservation (p=0.031 paired Student's t-Test). The unencapsulatedserogroup A strain F8239 (23) and the unencapsulated mynA mutant failedto colonize the mice at any time point, indicating a requirement ofserogroup A CPS in establishing colonization. Further, the mynCCAP+/OAc− mutant was impaired in the ability to maintain nasopharyngealcolonization when compared to the wild-type parent, suggesting thatO-acetylation of the serogroup A CPS may play a role in promotingpersistent meningococcal colonization.

The role of serogroup A CPS O-acetylation in protecting the meningococcifrom killing by pooled normal human sera was also investigated. In serumbactericidal activity assays (24), the wild-type parent and the mynCCAP+/OAc− mutant survived in 10% normal human sera (final concentration,v/v). In contrast, the unencapsulated mynA and mynB mutants of thisstrain were rapidly and completely killed (FIG. 9) by the same 10%normal human serum. In complement inactivated normal human serum (56°C., 30 min), both encapsulated and unencapsulated meningococci survived(grey bars). Both the CAP+/OAc+ wild-type and the CAP+/OAc− mynC mutantwere sensitive (>99% killing) to 25% and 50% (v/v) normal human sera.These results indicated that the O-acetylation did not enhance ordiminish the protection provided to meningococci by the serogroup Acapsule against complement-mediated bactericidal activity of normalhuman sera.

To investigate the role of O-acetylation of capsule in serogroup Ameningococcal vaccines, CPS of the CAP+/OAc+ wild-type parent and theCAP+/OAc− mynC mutant were prepared and standardized. These preparationswere used in inhibition ELISAs in which six well-standardized postvaccine sera (designated 242, 243, 268, 274, 414, 415) from serogroup Apolysaccharide vaccinated individuals (Menimmune®) were tested. OAc−serogroup A CPS competitively inhibited significantly less antibody thanthe wild-type CPS (FIG. 10A-10B) in five of the six sera tested. At thehighest concentrations of capsule used for inhibition (100 μg), OAc− CPSwas unable to inhibit one sera (274) did inhibit one serum (243), andonly incompletely inhibited (40-74%) four of the other sera (242, 268,414, 415). In contrast, the OAc+ CPS inhibited 75% to 100% of serumbactericidal activity of all six samples. These data confirmed theimportance of O-acetylation as a major epitope of the serogroup Ameningococcal polysaccharide-containing vaccines for most but not allindividuals.

The importance of serogroup A CPS O-3, O-4 acetylation as a factor inmeningococcal colonization, in resistance to killing by normal humansera and in serogroup A polysaccharide containing meningococcal vaccineswas addressed in this study. The identification of the serogroup A CPSO-acetyltransferase gene, mynC, has facilitated the generation of anencapsulated mutant devoid of O-acetylation. The OAc-mutant, its parentand other isogenic mutants of this strain permitted insights into therole of O-acetylation in serogroup A biology.

Colonization of the human nasopharynx is an essential step inmeningococcal pathogenesis (62). The complete failure to establishnasopharyngeal colonization of mice by serogroup A unencapsulatedmutants indicates a role of the serogroup A capsule in the initialpathogenic events after meningococcal acquisition in the upperrespiratory tract. A similar advantage in promoting nasopharyngealcolonization has been previously shown for the serogroup B capsule. Bothof these results may be correlated with a protective role of capsuleagainst elimination of meningococci by human host defenses at mucosalsurfaces at the time of initial acquisition. The impaired ability of themynC CAP+/OAc− to maintain colonization, compared to the wild-typeparent suggests an additional role of serogroup A capsule O-acetylationin meningococcal nasopharyngeal colonization of the nasopharynx. Thebulky O-acetyl groups might facilitate initial adherence interactionswith the nasopharyngeal mucosal epithelial surface or further enhanceresistance to mucosal host defenses. O-acetylation of theexopolysaccharide alginate in Pseudomonas aeruginosa has been shown tocontribute to biofilm and microcolony formation and to facilitateresistance to opsonic phagocytosis (35, 36). Also, O-acetylation ofrhizobial Nod factors defines host specificity and is critical for theformation of the preinfection thread and the root nodule inRhizobium-legume symbiosis (37, 38, 39).

The presence of capsule expressed on the surface of serogroup Ameningococci was important for protection against complement-mediatedbactericidal activity of normal human sera. Mutants such as mynA andmynB that lack CPS were rapidly killed by all concentrations of normalhuman sera. However, serogroup A capsule O-acetylation was not requiredfor or enhanced this protection. Both the encapsulated parent and OAc−mutant survived similarly in low concentrations of human sera. Lowconcentrations of sera (10% or less) are usually associated withantibody-mediated classical complement pathway activation (63) ratherthan alternative or possibly MBL lectin pathway activation requiringhigher concentrations of human sera.

Despite the disappearance of exposure to serogroup A N. meningitidis inthe United States and other industrialized countries, many individualsfrom these areas have serum bactericidal activity against serogroup Ameningococci. This may be due to antibodies directed at cross-reactiveserogroup A capsule-like epitopes present on Bacillus pumilus,Enterococcus faecalis and other commensal bacteria (64) or antibodies tononcapsular outer membrane epitopes, or components of the meningococcalsurface that lead to complement activation. In a recent study, Granoffand Amir (65) found a high prevalence of cross-reacting serogroup Acapsular antibodies in bactericidal sera from North America but only asmall number of these bactericidal sera were directly inhibited bypurified group A CPS. Complement activation by mannose-binding lectin(MBL) attachment to the Opa and PorB proteins of N. meningitidis hasbeen reported. Thus, in our study, serogroup A CPS, regardless of O-3 orO-4 acetylation, was protective against low levels of classical pathwaycomplement activation, but serogroup A meningococci, regardless ofO-acetylation, were killed by higher concentrations of normal humansera.

Berry et al. (8) previously studied the effects of N. meningitidisserogroup A capsular O-acetylation on development of immune responses toserogroup A CPS. Using chemical removal of O-acetyl groups, they foundthat a majority of antibodies generated by vaccination with serogroup ACPS were specific for epitopes involving O-acetyl groups and that adramatic reduction in immunogenicity was associated with removal ofthese groups. Similarly monoclonal antibodies against the O-acetylatedserotype 5 capsule of S. aureus are specific for the O-acetyl epitope(66). Among the meningococcal sialic acid capsules, serogroup Bmeningococci lack O-acetylation, serogroup C meningococci can expressO-7 or O-8 acetylation and serogroups W-135 and Y have variable O-7 orO-9 acetylation. In contrast to serogroup A meningococcal O-acetylation,O-acetylation of capsule in meningococcal serogroup C (32), pneumococcalserotype 9V (33) and E. coli K1 (67) do not appear essential for theinduction of protective antibodies.

Our data indicate that the O-acetyl group is a dominant epitope on theserogroup A CPS in individuals. Other capsular polysaccharides withimmuno-dominant O-acetyl epitopes are S. aureus serotype 5 (ManANAc O-3)and Salmonella typhi VI (GalANAc O-3). Interestingly, in capsules withimmuno-dominant acetylation epitopes, the acetylation sites are withinthe hexose sugar ring (endo-cyclic). In contrast, the O-acetyl epitopesof other capsules including the meningococcal sialic acid C, Y or W-135capsules (at positions O-7, O-8 or O-9), E. coli K1 (at positions O-7,O-9), and the S. pneumoniae serotype 9V capsule (ManNAc O-6) that do notcontribute to protective immunity are positioned in the exocyclic sidechain. Thus, the endo-cyclic position of O-acetylation, may have lessmobility compared to an exo-cyclic side chain location and appears toposition the O-acetyl group as a dominant epitope recognized by thehuman immune system.

In the present study, serogroup A O-acetylation did not enhance ordiminish resistance of meningococci to complement-mediated bactericidalactivity of normal human serum. However, O-acetylation of themeningococcal serogroup A CPS contributed in an animal model tonasopharyngeal colonization by this serogroup and was a major epitopefor antibodies generated by serogroup A CPS vaccination.

Expression refers to the transcription and translation of a structuralgene (coding sequence) so that a protein (i.e., expression product)having the biological activity of the O-acetyltransferase of the presentinvention is synthesized. It is understood that post-translationalmodification(s) in certain types of recombinant host cells may removeportions of the polypeptide which are not essential to enzymaticactivity.

The term expression control sequences refer to DNA sequences thatcontrol and regulate the transcription and translation of another DNAsequence (i.e., a coding sequence). A coding sequence is operativelylinked to an expression control sequence when the expression controlsequence controls and regulates the transcription and translation ofthat coding sequence. Expression control sequences include, but are notlimited to, promoters, enhancers, promoter-associated regulatorysequences, transcription termination and polyadenylation sequences, andtheir positioning and use is well understood by the ordinary skilledartisan. The term “operatively linked” includes having an appropriatestart signal (e.g., ATG) in front of the DNA sequence to be expressedand maintaining the correct reading frame to permit expression of theDNA sequence under the control of the expression control sequence andproduction of the desired product encoded by the DNA sequence. If a genethat one desires to insert into a recombinant DNA molecule does notcontain an appropriate start signal, such a start signal can be insertedin front of the gene. The combination of the expression controlsequences and the O-acetyltransferase coding sequence form theO-acetyltransferase expression cassette.

As used herein, an exogenous or heterologous nucleotide sequence is onewhich is not in nature covalently linked to a particular nucleotidesequence, e.g., an O-acetyltransferase coding sequence. Examples ofexogenous nucleotide sequences include, but are not limited to, plasmidvector sequences, expression control sequences not naturally associatedwith particular O-acetyltransferase coding sequences, and viral or othervector sequences. A non-naturally occurring DNA molecule is one whichdoes not occur in nature, and it is thus distinguished from achromosome, or example. As used herein, a non-naturally occurring DNAmolecule comprising a sequence encoding an expression product withO-acetyltransferase activity is one which comprises said coding sequenceand sequences which are not associated therewith in nature.

Similarly, as used herein an exogenous gene is one which does notnaturally occur in a particular recombinant host cell but has beenintroduced in using genetic engineering techniques well known in theart. An exogenous gene as used herein can comprise anO-acetyltransferase coding sequence expressed under the control of anexpression control sequence not associated in nature with said codingsequence.

Another feature of this invention is the expression of the sequencesencoding O-acetyltransferase. As is well-known in the art, DNA sequencesmay be expressed by operatively linking them to an expression controlsequence in an appropriate expression vector and employing thatexpression vector to transform an appropriate host cell.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,nonchromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., Escherichia coliplasmids colE1, pCR1, pBR322, pMB9 and their derivatives, plasmids suchas RP4; phage DNAs, e.g., M13 derivatives, the numerous derivatives ofphage λ, e.g., λgt11, and other phage DNA; yeast plasmids derived fromthe 2μ circle; vectors useful in eukaryotic cells, such as insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; baculovirus derivatives; and thelike. For mammalian cells there are a number of well-known expressionvectors available to the art.

Any of a wide variety of expression control sequences may be used inthese vectors to express the DNA sequences of this invention. Suchuseful expression control sequences include, for example, the early andlate promoters of SV40 or adenovirus for expression in mammalian cells,the lac system, the trp system, the tac or trc system, the majoroperator and promoter regions of phage λ, the control regions of fd coatprotein, the promoter for 3-phosphoglycerate kinase of phosphatase(e.g., pho5), the promoters of the yeast α-mating factors, and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Theskilled artisan understands which expression control sequences areappropriate to particular vectors and host cells.

A wide variety of host cells are also useful in expressing the DNAsequences of this invention. These hosts may include well-knownprokaryotic and eukaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as Chinese Hamster Ovary (CHO), R1.1, B-W and L-M cells,African Green Monkey kidney cells (e.g., COS 1, COS-7, BSC1, BSC40, andBMT10), insect cells (e.g., Sf9), and human cells and plant cells inculture.

It is understood that not all combinations of vector, expression controlsequence and host cell will function equally well to express the DNAsequences of this invention. However, one skilled in the art will beable to select the proper vector, expression control sequence, and hostcell combination without undue experimentation to accomplish the desiredexpression without departing from the scope of this invention.

In selecting a suitable expression control sequence, a variety offactors will normally be considered. These include, for example, therelative strength of the promoter, its controllability, and itscompatibility with the particular DNA sequence or gene to be expressed,e.g., with regard to potential secondary structure. Suitable hosts willbe selected by consideration of factors including compatibility with thechosen vector, secretion characteristics, ability to fold proteinscorrectly, and fermentation requirements, as well as any toxicity to thehost of the product encoded by the DNA sequences to be expressed, andthe ease of purification of the expression products. The practitionerwill be able to select the appropriate host cells and expressionmechanisms for a particular purpose.

Several strategies are available for the isolation and purification ofrecombinant O-acetyltransferase after expression in a host system. Onemethod involves expressing the proteins in bacterial cells, lysing thecells, and purifying the protein by conventional means. Alternatively,one can engineer the DNA sequences for secretion from cells. AnO-acetyltransferase protein can be readily engineered to facilitatepurification and/or immobilization to a solid support of choice. Forexample, a stretch of 6-8 histidines can be engineered throughpolymerase chain reaction or other recombinant DNA technology to allowpurification of expressed recombinant protein over a nickel-chargednitrilotriacetic acid (NTA) column using commercially availablematerials. Other oligopeptide “tags” which can be fused to a protein ofinterest by such techniques include, without limitation, strep-tag(Sigma-Genosys, The Woodlands, Tex.) which directs binding tostreptavidin or its derivative streptactin (Sigma-Genosys); aglutathione-S-transferase gene fusion system which directs binding toglutathione coupled to a solid support (Amersham Pharmacia Biotech,Uppsala, Sweden); a calmodulin-binding peptide fusion system whichallows purification using a calmodulin resin (Stratagene, La Jolla,Calif.); a maltose binding protein fusion system allowing binding to anamylose resin (New England Biolabs, Beverly, Mass.); and anoligo-histidine fusion peptide system which allows purification using aNi²⁺-NTA column (Qiagen, Valencia, Calif.).

Coding sequences which are synonymous to the coding sequence provided inSEQ ID NO:1 are within the scope of the present invention, as aresequences encoding O-acetyltransferases carrying out the same O-3 andO-4 acetylations of Neisseria meningitidis capsular polysaccharides, andwhere those sequences encode an O-acetyltransferases with at least 80%amino acid sequence identity with that of SEQ ID NO:2. All integersbetween 80 and 100% are included within the scope of the presentinvention in this context. In calculations of amino acid sequenceidentify, gaps inserted to optimize alignment are treated as mismatches.

O-acetyltransferase coding sequences from various N. meningitidisstrains have significant sequence homology to the exemplifiedO-acetyltransferase coding sequences, and the encoded enzymes have ahigh degree of amino acid sequence identity as disclosed herein. It isobvious to one normally skilled in the art that nonexemplified clonesand PCR amplification products can be readily isolated using standardprocedures and the sequence information provided herein. The ordinaryskilled artisan can utilize the exemplified sequences provided herein,or portions thereof, preferably at least 25-30 bases in length, inhybridization probes to identify cDNA (or genomic) clones encodingO-acetyltransferase, where there is at least 70% sequence homology tothe probe sequence using appropriate art-known hybridization techniques.The skilled artisan understands that the capacity of a cloned cDNA toencode functional O-acetyltransferase enzyme can be readily tested astaught herein.

Hybridization conditions appropriate for detecting various extents ofnucleotide sequence homology between probe and target sequences andtheoretical and practical consideration are given, for example in B. D.Hames and S. J. Higgins (1985) Nucleic Acid Hybridization, IRL Press,Oxford, and in Sambrook et al. (1989) supra. Under particularhybridization conditions the DNA sequences of this invention willhybridize to other DNA sequences having sufficient homology, includinghomologous sequences from different species. It is understood in the artthat the stringency of hybridization conditions is a factor in thedegree of homology required for hybridization. The skilled artisan knowshow to manipulate the hybridization conditions so that the stringency ofhybridization is at the desired level (high, medium, low). If attemptsto identify and isolate the O-acetyltransferase gene from another N.meningitidis strain fail using high stringency conditions, the skilledartisan will understand how to decrease the stringency of thehybridization conditions so that a sequence with a lower degree ofsequence homology will hybridize to the sequence used as a probe. Thechoice of the length and sequence of the probe is readily understood bythe skilled artisan.

The DNA sequences of this invention refer to DNA sequences prepared orisolated using recombinant DNA techniques. These include cDNA sequences,sequences isolated using PCR, DNA sequences isolated from their nativegenome, and synthetic DNA sequences. As used herein, this term is notintended to encompass naturally-occurring chromosomes or genomes. Thesesequences can be used to direct recombinant synthesis ofO-acetyltransferase for enzymatic acetylation of isolated capsularpolysaccharide, especially from N. meningitidis Serogroup A strains.

Isolated capsular polysaccharide is separated from the cells and culturemedium from which it was produced. Further purification is optional andwithin the realm of the skilled artisan.

In the present context, an in vitro enzymatic reaction, especially O-3and O-4 acetylation of Serogroup A N. meningitides capsularpolysaccharide, is carried out in the absence of whole, live cells. Theenzyme source can be a purified or partly purified enzyme or it can bepresent in a cell extract, recombinantly produced or otherwise, althoughgreater amounts per cell are produced through recombinant DNAtechnology.

It is well-known in the biological arts that certain amino acidsubstitutions can be made within a protein without affecting thefunctioning of that protein. Preferably such substitutions are of aminoacids similar in size and/or charge properties. For example, Dayhoff etal. (1978) in Atlas of Protein Sequence and Structure, Volume 5,Supplement 3, Chapter 22, pages 345-352, which is incorporated byreference herein, provides frequency tables for amino acid substitutionswhich can be employed as a measure of amino acid similarity. Dayhoff etal.'s frequency tables are based on comparisons of amino acid sequencesfor proteins having the same function from a variety of evolutionarilydifferent sources.

It will be a matter of routine experimentation for the ordinary skilledartisan to use the DNA sequence information presented herein to optimizeO-acetyltransferase expression in a particular expression vector andcell line for a desired purpose. A cell line genetically engineered tocontain and express an O-acetyltransferase coding sequence is useful forthe recombinant expression of protein products with the characteristicenzymatic activity of the specifically exemplified enzyme. Any meansknown to the art can be used to introduce an expressibleO-acetyltransferase coding sequence into a cell to produce a recombinanthost cell, i.e., to genetically engineer such a recombinant host cell.Recombinant host cell lines which express high levels ofO-acetyltransferase are useful as sources for the purification of thisenzyme, especially for in vitro acetylation of isolated capsularpolysaccharides, desirably those from N. meningitidis Serogroup A. Theamino acids which occur in the various amino acid sequences referred toin the specification have their usual three- and one-letterabbreviations routinely used in the art: A, Ala, Alanine; C, Cys,Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; F, Phe,Phenylalanine; G, Gly, Glycine; H, H is, Histidine; I, Ile, Isoleucine;K, Lys, Lysine; L, Leu, Leucine; M, Met, Methionine; N, Asn, Asparagine;P, Pro, Proline; Q, Gln, Glutamine; R, Arg, Arginine; S, Ser, Serine; T,Thr, Threonine; V, Val, Valine; W, Try, Tryptophan; Y, Tyr, Tyrosine.

A protein is considered an isolated protein if it is a protein isolatedfrom a host cell in which it is recombinantly produced. It can bepurified or it can simply be free of other proteins and biologicalmaterials with which it is associated in nature.

An isolated nucleic acid is a nucleic acid the structure of which is notidentical to that of any naturally occurring nucleic acid or to that ofany fragment of a naturally occurring genomic nucleic acid spanning morethan three separate genes. The term therefore covers, for example, a DNAwhich has the sequence of part of a naturally occurring genomic DNAmolecule but is not flanked by both of the coding or noncoding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and a recombinant nucleotide sequence that is partof a hybrid gene, i.e., a gene encoding a fusion protein. Specificallyexcluded from this definition are nucleic acids present in mixtures ofDNA molecules, transformed or transfected cells, and cell clones, e.g.,as these occur in a DNA library such as a cDNA or genomic DNA library.

In the present context, a promoter is a DNA region which includessequences sufficient to cause transcription of an associated(downstream) sequence. The promoter may be regulated, i.e., notconstitutively acting to cause transcription of the associated sequence.If inducible, there are sequences present which mediate regulation ofexpression so that the associated sequence is transcribed only when aninducer molecule is present in the medium in or on which the organism iscultivated.

One DNA portion or sequence is downstream of second DNA portion orsequence when it is located 3′ of the second sequence. One DNA portionor sequence is upstream of a second DNA portion or sequence when it islocated 5′ of that sequence.

One DNA molecule or sequence and another are heterologous to another ifthe two are not derived from the same ultimate natural source. Thesequences may be natural sequences, or at least one sequence can bedesigned by man, as in the case of a multiple cloning site region. Thetwo sequences can be derived from two different species or one sequencecan be produced by chemical synthesis provided that the nucleotidesequence of the synthesized portion was not derived from the sameorganism as the other sequence.

An isolated or substantially pure nucleic acid molecule orpolynucleotide is an O-acetyltransferase-encoding polynucleotide whichis substantially separated from other polynucleotide sequences whichnaturally accompany it on the N. meningitidis chromosome. The termembraces a polynucleotide sequence which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates, chemically synthesized analogues and analogues biologicallysynthesized by heterologous systems.

A polynucleotide is said to encode a polypeptide if, in its native stateor when manipulated by methods known to those skilled in the art, it canbe transcribed and/or translated to produce the polypeptide or afragment thereof. The anti-sense strand of such a polynucleotide is alsosaid to encode the sequence.

A nucleotide sequence is operably linked when it is placed into afunctional relationship with another nucleotide sequence. For instance,a promoter is operably linked to a coding sequence if the promotereffects its transcription or expression. Generally, operably linkedmeans that the sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in readingframe. However, it is well known that certain genetic elements, such asenhancers, may be operably linked even at a distance, i.e., even if notcontiguous.

The term recombinant polynucleotide refers to a polynucleotide which ismade by the combination of two otherwise separated segments of sequenceaccomplished by the artificial manipulation of isolated segments ofpolynucleotides by genetic engineering techniques or by chemicalsynthesis. In so doing one may join together polynucleotide segments ofdesired functions to generate a desired combination of functions.

Polynucleotide probes include an isolated polynucleotide attached to alabel or reporter molecule and may be used to identify and isolate otherO-acetyltransferase coding sequences, for example, those from othersstrains of N. meningitidis. Probes comprising synthetic oligonucleotidesor other polynucleotides may be derived from naturally occurring orrecombinant single or double stranded nucleic acids or be chemicallysynthesized. Polynucleotide probes may be labeled by any of the methodsknown in the art, e.g., random hexamer labeling, nick translation, orthe Klenow fill-in reaction, or with fluors or other detectablemoieties.

Large amounts of the polynucleotides may be produced by replication in asuitable host cell. Natural or synthetic DNA fragments coding for aprotein of interest are incorporated into recombinant polynucleotideconstructs, typically DNA constructs, capable of introduction into andreplication in a prokaryotic or eukaryotic cell, especially culturedmammalian cells, wherein protein expression is desired. Usually theconstruct is suitable for replication in a host cell, such as culturedmammalian cell or a bacterium, but a multicellular eukaryotic host mayalso be appropriate, with or without integration within the genome ofthe host cell. Commonly used prokaryotic hosts include strains ofEscherichia coli, although other prokaryotes, such as Bacillus subtilisor a pseudomonad, may also be used. Eukaryotic host cells includemammalian cells, yeast, filamentous fungi, plant, insect, amphibian andavian cell lines. Such factors as ease of manipulation, ability toappropriately glycosylate expressed proteins, degree and control ofrecombinant protein expression, ease of purification of expressedproteins away from cellular contaminants or other factors influence thechoice of the host cell.

The polynucleotides may also be produced by chemical synthesis, e.g., bythe phosphoramidite method described by Beaucage and Caruthers (1981)Tetra. Letts. 22: 1859-1862 or the triester method according to Matteuciet al. (1981) J. Am. Chem. Soc. 103: 3185, and may be performed oncommercial automated oligonucleotide synthesizers. A double-strandedfragment may be obtained from the single stranded product of chemicalsynthesis either by synthesizing the complementary strand and annealingthe strand together under appropriate conditions or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence. DNA constructs prepared for introduction into a prokaryotic oreukaryotic host will typically comprise a replication system (i.e.vector) recognized by the host, including the intended DNA fragmentencoding the desired polypeptide, and will preferably also includetranscription and translational initiation regulatory sequences operablylinked to the polypeptide-encoding segment. Expression systems(expression vectors) may include, for example, an origin of replicationor autonomously replicating sequence (ARS) and expression controlsequences, a promoter, an enhancer and necessary processing informationsites, such as ribosome-binding sites, RNA splice sites, polyadenylationsites, transcriptional terminator sequences, and mRNA stabilizingsequences. Signal peptides may also be included where appropriate fromsecreted polypeptides of the same or related species, which allow theprotein to cross and/or lodge in cell membranes or be secreted from thecell.

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host. Examples of workablecombinations of cell lines and expression vectors are described inSambrook et al. (1989) vide infra; Ausubel et al. (Eds.) (1995) CurrentProtocols in Molecular Biology, Greene Publishing and WileyInterscience, New York; and Metzger et al. (1988) Nature, 334: 31-36.Many useful vectors for expression in bacteria, yeast, fungal,mammalian, insect, plant or other cells are well known in the art andmay be obtained from such vendors as Stratagene, New England Biolabs,Promega Biotech, and others. In addition, the construct may be joined toan amplifiable gene (e.g., DHFR) so that multiple copies of the gene maybe made. For appropriate enhancer and other expression controlsequences, see also Enhancers and Eukaryotic Gene Expression, ColdSpring Harbor Press, N.Y. (1983). While such expression vectors mayreplicate autonomously, they may less preferably replicate by beinginserted into the genome of the host cell.

Expression and cloning vectors will likely contain a selectable marker,that is, a gene encoding a protein necessary for the survival or growthof a host cell transformed with the vector. Although such a marker genemay be carried on another polynucleotide sequence co-introduced into thehost cell, it is most often contained on the cloning vector. Only thosehost cells into which the marker gene has been introduced will surviveand/or grow under selective conditions. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxicsubstances, e.g., ampicillin, neomycin, methotrexate, etc.; (b)complement auxotrophic deficiencies; or (c) supply critical nutrientsnot available from complex media. The choice of the proper selectablemarker will depend on the host cell; appropriate markers for differenthosts are known in the art.

Recombinant host cells, in the present context, are those which havebeen genetically modified to contain an isolated DNA molecule of theinstant invention. The DNA can be introduced by any means known to theart which is appropriate for the particular type of cell, includingwithout limitation, transfection, transformation, lipofection orelectroporation.

It is recognized by those skilled in the art that the DNA sequences mayvary due to the degeneracy of the genetic code and codon usage. All(synonymous) DNA sequences which code for the O-acetyltransferaseprotein are included in this invention, including the DNA sequence asgiven in FIG. 8A. Also contemplated are coding sequences which encode anO-acetyltransferase as taught herein with at least 80% amino acidsequence identity to that of SEQ ID NO:2.

Additionally, it will be recognized by those skilled in the art thatallelic variations may occur in the DNA sequences which will notsignificantly change activity of the amino acid sequences of thepeptides which the DNA sequences encode. All such equivalent DNAsequences are included within the scope of this invention and thedefinition of the regulated promoter region. The skilled artisan willunderstand that the sequence of the exemplified O-acetyltransferaseprotein and the nucleotide sequence encoding it can be used to identifyand isolate additional, nonexemplified nucleotide sequences which arefunctionally equivalent to the sequences given FIG. 8A.

Hybridization procedures are useful for identifying polynucleotides withsufficient homology to the subject coding sequence to be useful astaught herein. The particular hybridization technique is not essentialto the subject invention. As improvements are made in hybridizationtechniques, they can be readily applied by one of ordinary skill in theart.

A probe and sample are combined in a hybridization buffer solution andheld at an appropriate temperature until annealing occurs. Thereafter,the membrane is washed free of extraneous materials, leaving the sampleand bound probe molecules typically detected and quantified byautoradiography and/or liquid scintillation counting. As is well knownin the art, if the probe molecule and nucleic acid sample hybridize byforming a strong non-covalent bond between the two molecules, it can bereasonably assumed that the probe and sample are essentially identical,or completely complementary if the annealing and washing steps arecarried out under conditions of high stringency. The probe's detectablelabel provides a means for determining whether hybridization hasoccurred.

In the use of the oligonucleotides or polynucleotides as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³²P, ³⁵S, or the like.Non-radioactive labels include, for example, ligands such as biotin orthyroxine, as well as enzymes such as hydrolases or peroxidases, or achemiluminescent reagent such as luciferin, or fluorescent compoundslike fluorescein and its derivatives. Alternatively, the probes can bemade inherently fluorescent as described in International ApplicationNo. WO 93/16094.

Various degrees of stringency of hybridization can be employed. The morestringent the conditions, the greater the complementarity that isrequired for duplex formation. Stringency can be controlled bytemperature, probe concentration, probe length, ionic strength, time,and the like. Preferably, hybridization is conducted under moderate tohigh stringency conditions by techniques well know in the art, asdescribed, for example in Keller, G. H., M. M. Manak (1987) DNA Probes,Stockton Press, New York, N.Y., pp. 169-170, hereby incorporated byreference.

As used herein, moderate to high stringency conditions for hybridizationare conditions which are particularly advantageous. An example of highstringency conditions are hybridizing at 68° C. in 5×SSC/5×Denhardt'ssolution/0.1% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature.An example of conditions of moderate stringency are hybridizing at 68°C. in 5×SSC/5×Denhardt's solution/0.1% SDS and washing at 42° C. in3×SSC. The parameters of temperature and salt concentration can bevaried to achieve the desired level of sequence identity between probeand target nucleic acid. See, e.g., Sambrook et al. (1989) vide infra orAusubel et al. (1995) Current Protocols in Molecular Biology, John Wiley& Sons, NY, N.Y., for further guidance on hybridization conditions.

Specifically, hybridization of immobilized DNA in Southern blots with³²P-labeled gene specific probes is performed according to standardmethods (Maniatis et al.) In general, hybridization and subsequentwashes were carried out under moderate to high stringency conditionsthat allowed for detection of target sequences with homology to theexemplified sequence. For double-stranded DNA gene probes, hybridizationcan be carried out overnight at 20-25° C. below the melting temperature(Tm) of the DNA hybrid in 6×SSPE 5×Denhardt's solution, 0.1% SDS, 0.1mg/ml denatured DNA. The melting temperature is described by thefollowing formula (Beltz, G. A., Jacobe, T. H., Rickbush, P. T.,Chorbas, and F. C. Kafatos (1983) Methods of Enzymology, R. Wu, L,Grossman and K Moldave (eds) Academic Press, New York 100:266-285).Tm=81.5° C.+16.6 Log [Na+]+0.41(+G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows: twice at room temperaturefor 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash), and once atTM-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringencywash).

For oligonucleotide probes, hybridization is carried out overnight at10-20° C. below the melting temperature (Tm) of the hybrid 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes is determined by the following formula: TM(°C.)=2(number T/A base pairs+4(number G/C base pairs) (Suggs, S. V. etal. (1981) ICB-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown(ed.), Academic Press, New York, 23:683-693).

Washes are typically carried out as follows: twice at room temperaturefor 15 minutes 1×SSPE, 0.1% SDS (low stringency wash), and once at thehybridization temperature for 15 minutes in 1×SSPE, 0.1% SDS (moderatestringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment>70 or so bases in length, the followingconditions can be used: Low, 1 or 2×SSPE, room temperature; Low, 1 or2×SSPE, 42° C.; Moderate, 0.2× or 1×SSPE, 65° C.; and High, 0.1×SSPE,65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, and those methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

Thus, mutational, insertional, and deletional variants of the disclosednucleotide sequences can be readily prepared by methods which are wellknown to those skilled in the art. These variants can be used in thesame manner as the exemplified primer sequences so long as the variantshave substantial sequence homology with the original sequence. As usedherein, substantial sequence homology refers to homology which issufficient to enable the variant polynucleotide to function in the samecapacity as the polynucleotide from which the probe was derived.Preferably, this homology is greater than 80%, more preferably, thishomology is greater than 85%, even more preferably this homology isgreater than 90%, and most preferably, this homology is greater than95%. The degree of homology or identity needed for the variant tofunction in its intended capacity depends upon the intended use of thesequence. It is well within the skill of a person trained in this art tomake mutational, insertional, and deletional mutations which areequivalent in function or are designed to improve the function of thesequence or otherwise provide a methodological advantage.

Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primedsynthesis of a nucleic acid sequence. This procedure is well known andcommonly used by those skilled in this art (see, e.g., Mullis, U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science230:1350-1354). PCR is based on the enzymatic amplification of a DNAfragment of interest that is flanked by two oligonucleotide primers thathybridize to opposite strands of the target sequence. The primers areoriented with the 3′ ends pointing towards each other. Repeated cyclesof heat denaturation of the template, annealing of the primers to theircomplementary sequences, and extension of the annealed primers with aDNA polymerase result in the amplification of the segment defined by the5′ ends of the PCR primers. Since the extension product of each primercan serve as a template for the other primer, each cycle essentiallydoubles the amount of DNA template produced in the previous cycle. Thisresults in the exponential accumulation of the specific target fragment,up to several million-fold in a few hours. By using a thermostable DNApolymerase such as the Taq polymerase, which is isolated from thethermophilic bacterium Thermus aquaticus, the amplification process canbe completely automated. Other enzymes which can be used are known tothose skilled in the art.

It is well known in the art that the polynucleotide sequences of thepresent invention can be truncated and/or mutated such that certain ofthe resulting fragments and/or mutants of the original full-lengthsequence can retain the desired characteristics of the full-lengthsequence. A wide variety of restriction enzymes which are suitable forgenerating fragments from larger nucleic acid molecules are well known.In addition, it is well known that Bal31 exonuclease can be convenientlyused for time-controlled limited digestion of DNA. See, for example,Maniatis (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, pages 135-139, incorporated herein byreference. See also Wei et al. (1983 J. Biol. Chem. 258:13006-13512. Byuse of Bal31 exonuclease (commonly referred to as “erase-a-base”procedures), the ordinarily skilled artisan can remove nucleotides fromeither or both ends of the subject nucleic acids to generate a widespectrum of fragments which are functionally equivalent to the subjectnucleotide sequences. One of ordinary skill in the art can, in thismanner, generate hundreds of fragments of controlled, varying lengthsfrom locations all along the original O-acetyltransferase codingsequence. The ordinarily skilled artisan can routinely test or screenthe generated fragments for their characteristics and determine theutility of the fragments as taught herein. It is also well known thatthe mutant sequences of the full length sequence, or fragments thereof,can be easily produced with site directed mutagenesis. See, for example,Larionov, O. A. and Nikiforov, V. G. (1982) Genetika 18(3):349-59;Shortle, D, DiMaio, D., and Nathans, D. (1981) Annu. Rev. Genet.15:265-94; both incorporated herein by reference. The skilled artisancan routinely produce deletion-, insertion-, or substitution-typemutations and identify those resulting mutants which contain the desiredcharacteristics of the full length wild-type sequence, or fragmentsthereof, i.e., those which retain O-acetyltransferase activity asdetermined herein.

DNA sequences having at least 80, 90, or at least 95% (and all integersand ranges between 80 and 100%) identity to the recited DNA sequence ofFIG. 8A and functioning to encode an O-acetyltransferase protein arewithin the scope of this invention. Such functional equivalents areincluded in the definition of an O-acetyltransferase coding sequence.Following the teachings herein and using knowledge and techniques wellknown in the art, the skilled worker will be able to make a large numberof operative embodiments having equivalent DNA sequences to those listedherein without the expense of undue experimentation.

As used herein percent sequence identity of two nucleic acids isdetermined using the algorithm of Altschul et al. (1997) Nucl. AcidsRes. 25: 3389-3402; see also Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches areperformed with the NBLAST program, score=100, wordlength=12, to obtainnucleotide sequences with the desired percent sequence identity. Toobtain gapped alignments for comparison purposes, Gapped BLAST is usedas described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (NBLAST and XBLAST) are used. See theNational Center for Biotechnology Information on the internet.

In another embodiment, immunogenic compositions for producing polyclonaland/or monoclonal antibodies capable of specifically binding toO-acetyltransferase, O-3 and or O-4 acetylated capsular polysaccharidefrom N. meningitidis, especially Serogroup A, (or fragments thereof) areprovided. The term antibody is used to refer both to a homogenousmolecular entity and a mixture such as a serum product made up of aplurality of different molecular entities. Monoclonal or polyclonalantibodies, preferably monoclonal, specifically reacting with aparticular epitope in a molecule of interest may be made by methodsknown in the art. See, e.g., Harlow and Lane (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratories; Goding (1986)Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press,New York; and Ausubel et al. (1993) supra. Also, recombinantimmunoglobulins may be produced by methods known in the art, includingbut not limited to, the methods described in U.S. Pat. No. 4,816,567,incorporated by reference herein. Monoclonal antibodies with affinitiesof 10⁸ M⁻¹, preferably 10⁹ to 10¹⁰ or more are preferred.

Antibodies generated against a molecule of interest are useful, forexample, as probes for screening DNA expression libraries or fordetecting the presence of particular neisserial strains or theirisolated capsular polysaccharides in a test sample. Hydrophilic regionsof the O-acetyltransferase of the present invention can be identified bythe skilled artisan, and peptide antigens can be synthesized andconjugated to a suitable carrier protein (e.g., bovine serum albumin orkeyhole limpet hemocyanin) for use in vaccines or in raising antibodyspecific for LOS biosynthetic proteins. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornoncovalently, a substance which provides a detectable signal. Suitablelabels include but are not limited to radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent agents, chemiluminescentagents, magnetic particles and the like. United States patentsdescribing the use of such labels include but are not limited to U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241.

Antibodies specific for the O-3 and/or O-4 acetylated capsularpolysaccharide from N. meningitidis are useful in preventing diseaseresulting from neisseriae, especially N. meningitidis infections. Suchantibodies can be obtained by the methods described above. Because thereis some loss of acetyl residues during isolation of the capsularpolysaccharide and because there is some loss of immunogenicity of anunacetylated or a poorly acetylated capsular polysaccharide, the qualityof a N. meningitidis capsular polysaccharide-containing immunogeniccomposition, it is advantageous to treat a capsular polysaccharidepreparation with the O-acetyltransferase of the present invention priorto use in immunogenic compositions, including vaccine compositions.

Compositions and immunogenic preparations, including vaccinecompositions comprising in vitro acetylated capsular polysaccharide fromN. meningitidis, especially Serogroup A N. meningitides, and a suitablecarrier therefor are provided. Immunogenic compositions are those whichresult in specific antibody production when injected into a human or ananimal. Such immunogenic compositions are useful, for example, inimmunizing a human, against infection by neisserial pathogenic strains,especially those of Serogroup A N. meningitidis. The immunogenicpreparations comprise an immunogenic amount of an in vitro acetylatedcapsular polysaccharide preparation derived from a N. meningitidisstrain, especially Serogroup A, and a suitable carrier.

The immunogenic compositions advantageously further compriselipooligosaccharide(s), proteins and/or neisserial cells of Serogroup AN. meningitidis and optionally, one or more other serological types,including but not limited to any known to the art. It is understand thatwhere whole cells are formulated into the immunogenic composition, thecells are preferably inactivated, especially if the cells are of avirulent strain. Such immunogenic compositions may comprise one or moreLOS preparations, or another protein or other immunogenic cellularcomponent. By “immunogenic amount” is meant an amount capable ofeliciting the production of antibodies directed against neisserialcapsular polysaccharides in an animal or human to which the vaccine orimmunogenic composition has been administered.

Immunogenic carriers may be used to enhance the immunogenicity of acomponent of the immunogenic composition as known to the art. Suchcarriers include, but are not limited to, proteins and polysaccharides,liposomes, and bacterial cells and membranes. Protein carriers may bejoined to the molecule(s) of interest to form fusion proteins byrecombinant or synthetic means or by chemical coupling. Useful carriersand means of coupling such carriers to polypeptide antigens are known inthe art. The art knows how to administer immunogenic compositions so asto generate protective immunity on the mucosal surfaces of the upperrespiratory system, especially the mucosal epithelium of thenasopharynx, where immunity is specific for N. meningitidis, as well asprotecting other parts of the body.

The immunogenic compositions of the present invention may be formulatedby any of the means known in the art. Such vaccines are typicallyprepared as injectables, either as liquid solutions or suspensions.Solid forms suitable for solution in, or suspension in, liquid prior toinjection may also be prepared. The preparation may also, for example,be emulsified, or the protein encapsulated in liposomes.

The active immunogenic ingredients are often mixed with excipients orcarriers which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients include but are not limited towater, saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. The concentration of the immunogenic polypeptide in injectableformulations is usually in the range of 0.2 to 5 mg/ml.

In addition, if desired, the vaccines may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutamMyl-L-alanine-2-(1′-2′-dipahnitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A referred to as MTP-PE); and RIM, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN80 emulsion (TWEEN 80 is polyoxyethylene sorbitan monooleate). Theeffectiveness of an adjuvant may be determined by measuring the amountof antibodies directed against the immunogen resulting fromadministration of the immunogen in vaccines which are also comprised ofthe various adjuvants. Such additional formulations and modes ofadministration as are known in the art may also be used.

In vitro acetylated capsular polysaccharide from N. meningitidis andadvantageously containing cells of N. meningitidis may be formulatedinto immunogenic compositions as neutral or salt forms. Preferably, whencells are used they are of attenuated or avirulent strains, or the cellsare killed before use. Pharmaceutically acceptable salts include but arenot limited to the acid addition salts (formed with free amino groups ofthe peptide) which are formed with inorganic acids, e.g., hydrochloricacid or phosphoric acids; and organic acids, e.g., acetic, oxalic,tartaric, or maleic acid. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases, e.g., sodium, potassium, ammonium,calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine,trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.

The immunogenic preparations of the present invention are administeredin a manner compatible with the dosage formulation, and in such amountas will be prophylactically and/or therapeutically effective. Thequantity to be administered, which is generally in the range of about100 to 1,000 μg of in vitro acetylated polysaccharide per dose, moregenerally in the range of about 1 to 500 μg per dose, depends on thesubject to be treated, the capacity of the individual's immune system tosynthesize antibodies, and the degree of protection desired. Preciseamounts of the active ingredient required to be administered may dependon the judgment of the physician and may be peculiar to each individual,but such a determination is within the skill of such a practitioner.

The vaccine or other immunogenic composition may be given in a singledose or multiple dose schedule. A multiple dose schedule is one in whicha primary course of vaccination may include 1 to 10 or more separatedoses, followed by other doses administered at subsequent time intervalsas required to maintain and or reinforce the immune response, e.g., at 1to 4 months for a second dose, and if needed, a subsequent dose(s) afterseveral months.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventiondescribed herein may be practiced in the absence of any element orelements, limitation or limitations which is not specifically disclosedherein, provided that there would be no anticipation by or obviousnessover prior art.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention. Changes therein and other uses will occur to thoseskilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and all references cited herein are herebyincorporated by reference to the extent that there is no inconsistencywith the present specification.

Monoclonal or polyclonal antibodies, preferably monoclonal, specificallyreacting with a polypeptide or protein of interest may be made bymethods known in the art. See, e.g., Harlow and Lane (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratories; Goding (1986)Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press,New York; and Ausubel et al. (1993) Current Protocols in MolecularBiology, Wiley Interscience, New York, N.Y.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York; and Ausubel et al. (1992) Current Protocols in MolecularBiology, Greene/Wiley, New York, N.Y. Abbreviations and nomenclature,where employed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein.

The specifically exemplified compounds and methods and accessory methodsdescribed herein are representative of particular embodiments of thepresent invention; they are not intended to limit the scope of theinvention. Thus, additional embodiments are within the scope of theinvention and within the following claims.

EXAMPLES Example 1 Materials and Bacterial Strains

Bacterial strains, plasmids and primers used in this study are describedin Table 1. The serogroup A meningococcal strains were originallyisolated during an outbreak in Nairobi, Kenya in 1989 (12) and wereprovided by the Centers for Disease Control and Prevention (CDC),Atlanta, Ga. Strain F8229 (CDC1750) is encapsulated and was isolatedfrom the cerebrospinal fluid of a patient with meningitis. Strain F8239(CDC16N3) is an unencapsulated variant originally isolated as aserogroup A strain from the pharynx of an asymptomatic carrier. Thesestrains belong to clonal group III-II and are closely related to strainsthat have caused epidemics in Saudi Arabia, Chad, Ethiopia and otherparts of the world.

Monoclonal antibody 14-1-A (13) against meningococcal serogroup Acapsular polysaccharide was provided by Dr. Wendell Zollinger, WalterReed Army Institute of Research.

Restriction enzymes were purchased from New England Biolabs (Beverly,Mass.). Ni-NTA agarose gravity flow matrix and the Anti-Penta-Hismonoclonal antibodies were purchased from Qiagen Inc. (Valencia,Calif.). The B-PER 6X-His Fusion protein purification kit was purchasedfrom Pierce (Rockford, Ill.). ¹⁴C-labeled acetyl coenzyme A waspurchased from Sigma (St. Louis, Mo.). Automated DNA sequence analysiswas performed with the Prism Dye-Deoxy Terminator Cycle Sequencing Kit(Applied Biosystems, Foster City, Calif.), and completed reactions wererun on an ABI model 377 automated DNA sequencer.

Example 2 Growth Conditions

Meningococcal strains were grown with 3.5% CO2 at 37° C. on GC base agar(Difco, Detroit, Mich.), supplemented with 0.4% glucose and 0.68 mM Fe(NO3)3, or in GC broth containing the same supplements and 0.043%NaHCO3. BHI medium (37 g/liter brain heart infusion) with 1.25% fetalbovine serum was used when kanamycin selection was required. Antibioticconcentrations (in μg/ml) used for E. coli strains were ampicillin, 100,kanamycin, 50, and erythromycin, 300; and for N. meningitidis werekanamycin, 80, spectinomycin, 60, erythromycin, 3. E. coli DH5α strain,cultured on Luria Bertani (LB) medium, was used for cloning andpropagation of plasmids. Meningococci were transformed by the procedureof Janik et al. (14). E. coli strains were transformed byelectroporation (Gene-pulser Bio-Rad, Hercules, Calif., according to themanufacturer's protocol).

Example 3 Construction of Meningococcal mynC Nonpolar Mutant NmA001

An internal 745-bp fragment of mynC, produced by PCR amplification usingprimers SE57 and SE61 (11) and the chromosomal DNA of strain F8229 as atemplate, was cloned into pCR2.1 to yield pGS201. The aphA-3 fragmentobtained from pUC18K with EcoR I and HinC II digestion and filled inwith Klenow polymerase was inserted into the unique SspI site of mynC inpGS201 to generate pGS202. The correct orientation of aphA-3 wasconfirmed by colony PCR and direct sequencing analysis of pGS202. AScaI-linearized pGS202 plasmid was used to transform serogroup Ameningococcal strain F8229 to generate NmA001. The correct homologousrecombination of the aphA-3 cassette into the mynC coding sequence wasconfirmed by PCR with cassette-specific primers and chromosomal-specificprimers.

Example 4 Overexpression and Purification of Meningococcal MynC

The complete coding sequence of mynC was obtained by PCR amplificationusing SG005 (NdeI) and SG006 (XhoI) primers (Table 1). The PCR product,digested with NdeI and XhoI, was subsequently cloned into pET20b(+) cutwith the same enzymes to yield pGS203 that resulted in a C-terminal(His)₆ fusion. pGS203 plasmid was purified and subjected to DNA sequenceanalysis to confirm the intact mynC sequence and the C-terminal His tagfusion. pGS203 was then transformed into the E. Coli expression strainBLR21 (DE3) pLysS. One liter of LB culture of the MynC overexpressionstrain was induced with 1 mM IPTG for 5 h. The harvested cells wereresuspended in 15 ml of lysis buffer (50 mM sodium phosphate, pH 8.0;300 mM NaCI; 10 mM imidazole; 1% (v/v final concentration) TWEEN 20(TWEEN 20 is polyoxyethylene sorbitan monolaurate), 1 mMphenylmethylsulfonyl fluoride and 1 mg/ml lysozyme) left on ice for 30min and sonicated 10 times for 30 s with 30 s cooling intervals. Thecell debris was removed by centrifugation at 14,000×g for 15 min at 4°C. The over-expressed protein was purified under native conditions onNi-NTA (nickel-nitrilotriacetic acid) (Qiagen) matrices following thesupplier's protocol with modification in column washing. Briefly, thecrude extract was incubated with 2 ml of 50% suspension of Ni-NTAagarose for 1 h before packing into a column. The column was washed with5 ml each of 10, 20 and 40 mM imidazole in lysis buffer (wash 1, wash 2and wash 3, respectively) and then eluted with 5 ml of 250 mM imidazolecontaining buffer. The MynC protein was also extracted and purified,using a Pierce B-PER protein extraction kit (15), containing a lysisreagent with a mild nonionic detergent in 20 mM Tris.HCI (pH 7.5),following the manufacturer's instructions. The purified MynC fractionsof either methods were concentrated separately by Centricon YM-3centrifugal filters (Millipore Corporation, Bedford, Mass.) afterSDS-PAGE analysis and dialyzed in storage buffer (50 mM HEPES, pH 7.05,5 mM MgCl₂, 100 mM NaCI and 1 mM EDTA). The protein concentration wasdetermined with BCA protein assay kit (Pierce, Rockford, Ill.) using BSAas standard.

Example 5 Complementation of the NmA001 Mutant

An intact copy of mynC coding sequence under the control of the tacpromoter was constructed on a meningococcal shuttle vector. Full-lengthmynC with a C-terminal His tag was amplified from pGS203 using primersSG007 (HindIII) and SG008 (EcoRI) (Table 1). The amplified PCR productwas cloned in pCR 2.1 to yield pGS204. The mynC insert was subsequentlyreleased from pGS204 with HindIII and EcoRV digestion and ligated intothe HindIII and SmaI sites of pFlag-CTC to generate pGS205, with mynCunder the control of the lac promoter. The construct was confirmed byPCR using YT79 and YT80 vector-specific primers. The pGS205 plasmid wasthen cut with BglI, filled in with Klenow, and ligated into the EcoRVsite of the meningococcal shuttle vector, pYT250 (Erm^(R)), yieldingpGS206. The pGS206 construct was methylated with HaeIII methylase andthe reaction mixture used directly to transform the wild type strainF8229 and the mynC nonpolar mutant NmA001, yielding NmAwtc1 and NmAnpc1,respectively.

Example 6 Meningococcal Membrane and Cytosolic Preparations

Meningococcal membranes and cytosols were separated by the method ofClark et al. (16) from the mynC-complemented meningococcal strainNmAnpc1. Briefly, the 500 ml culture pellet of NmAnpc1 carrying pGS205(mynC), induced overnight with 1 mM IPTG, was used to produce the innerand outer membrane and cytosol preparations. The pellet was suspended in2 ml of lysis buffer (1 mM EDTA, 50 mM Tris, 20% sucrose, pH 8.0 with 1mg/ml lysozyme) and incubated for 30 min at 4° C. Spheroplasts werediluted with 20 ml Tris buffer and were sonicated for three times, eachfor 30 seconds, in an ice bath with 30 second resting intervals. Thecell debris was removed by centrifuging at 10K for 15 min at 4° C. Thesupernatant was freeze-thawed once at −70° C. before ultracentrifugationat 100,000×g for 90 min at 4° C. The pellet, containing themeningococcal membrane fraction, was washed with Tris buffer. The levelof contamination of membrane fraction with cytoplasmic components wasassessed by determining the activity of the cytoplasmic enzyme malatedehydrogenase (17) for both fractions. The membrane fractions were97-98% pure. The cytosolic proteins were precipitated using 5%trichloroacetic acid and suspended in 2 ml of 1 M Tris (pH 6.8). Totalmembrane was solubilized with 2 ml of 2% N-lauroylsarcosine (sarcosyl)in 10 mM HEPES buffer pH 7.4 and stabilized for 1 h at room temperatureusing an orbital shaker.

Soluble inner membrane components and insoluble outer membranecomponents were separated by ultracentrifugation at 100,000×g for 2 h at4° C. The outer membrane pellet was suspended in 500 μl of 1M Tris (pH6.8). The diluted inner membrane components were precipitated using 5%trichloroacetic acid, and the pellet thus obtained was suspended in 500μl of 1M Tris (pH 6.8). Sub-cellular fractions were loaded on PAGE gelsbased on a set amount of starting 500 ml cell culture pellet (˜1×10¹¹cells) and analyzed by western blots.

Membrane solubilization experiments were performed as described (18).Briefly, the membrane pellets were extracted with 5 ml of phosphatebuffer (pH 7.6) containing 0.2 mM dithiothreitol, 20% sucrose, 0.2 MKCl, and either 1% Triton X-100, 1 M NaCl, or 6 M urea for 30 min atroom temperature (urea), at 30° C. (Tx-100), or on ice (buffer alone,buffer with NaCl). Samples were centrifuged for 1 h at 130,000×g (4° C.)after the extraction. Proteins in the soluble fractions wereprecipitated using 5% trichloroacetic acid, and the precipitatesobtained were washed two times in acetone, dried and re-suspended in 1MTris (pH 6.8) before an equal volume of 2×SDS-PAGE sample buffer wasadded.

Example 7 CPS Extraction and Structural Characterization

Capsular polysaccharide was extracted from two liters of meningococcalcultures using the method of Gotschlich et al. (19). Briefly, theovernight cultures were treated with a final concentration of 1%CETLAVLON, a polycationic detergent that precipitates the polyanionicpolysaccharides. The precipitate was collected by centrifugation andresuspended in water, and CaCl₂ was then added to a final concentrationof 1 mM in order to separate the polysaccharide from the detergent.Nucleic acids were precipitated from the solution by adding 25% (v/v) ofethanol followed by centrifugation. CPS in the supernatant wassubsequently precipitated using ethanol at a final concentration of 80%(v/v). Contaminating protein, traces of CETAVLON (polycationicdetergent) and other low molecular weight contaminants were removed withproteinase K digestion and extensive dialysis against a buffer composedof 10% ethanol, 50 mM NaCl, 5 mM Tris. CPS was further purified using aSEPHACRYL 200 (gel filtration) column with 50 mM ammonium formateelution. Column fractions were tested for neutral sugar using the phenolsulfuric acid assay (20). Void volume fractions were pooled andconcentrated by speed vacuuming and analyzed by DOC-PAGE and Alcian bluestaining (21).

Example 8 Compositional and NMR Analysis of Capsular Polysaccharides

Compositional analysis of purified CPS was performed on the alditolacetate derivatives of the sugars after removal of the phosphate groupsby the HF treatment of the purified NmA CPS. The alditol acetatederivatives were analyzed by the combined gas chromatography/massspectrometry using 30-m SP2330 capillary column (Supelco) (22).

Lyophilized wild type or mutant capsular polysaccharide powder (5 mg)was dissolved in D₂O (Sigma, 99.999% atom D) to a uniform concentrationof 5 mg/ml. Solutions were agitated by vortexing for 10 minutes at roomtemperature. Low speed centrifugation (7200×g for 10 min) removedundissolved material. Aliquots (600 μl) of the supernatant weretransferred to 5 mm NMR tubes and placed in a sonication bath for 10minutes to eliminate air bubbles trapped on the inner wall of the NMRtubes.

NMR spectra were acquired on a Varian Unity 500 NMR spectrometerequipped with a 5 mm PFG triple resonance probe, high precisiontemperature controller (+0.1° C.), and under the control of VNMR version6.1B, or a Varian Inova 500 spectrometer equipped with a 5 mm PFGinverse detection hetero nuclear probe, running under VNMR version 6.1Cand Solaris 2.8. One-dimensional (1-D) proton NMR spectra were collectedat 25° C. using a standard one-pulse experiment. The transmitter was setat the HDO frequency (4.78 ppm). Standard spectral acquisitionconditions are to collect 64 K data points over a spectral window of8000 Hz. The acquisition time is 4.096 s and a relaxation delay of 26 sis included, giving a recycle time of 30 s. Typically, 64 scans wereaveraged. Spectra were Fourier-transformed after applying a 0.2 Hz linebroadening function. Integrations were performed using subroutines builtinto the VNMR software.

Example 9 Hydrophobic Interaction Chromatography

The cell surface hydrophobicity of meningococcal strains was testedusing a modified method of Field et al. (23). Disposable plastic columnspacked with octyl agarose (Sepharose CL-4B, Sigma) to a height of 2 cmwere washed with 10 ml of Buffer A (0.2 M ammonium sulphate in 10 mMsodium phosphate buffer, pH 6.8). Meningococci collected from overnightplate cultures were suspended in phosphate, buffered saline (PBS) to anoptical density of 10, and a 100 μl aliquot was gently pipetted onto thesurface of the column and eluted with 5 ml Buffer A. A 100 μl cellsuspension diluted directly into 5 ml of Buffer A was also prepared as acontrol. The OD₆₀₀ values of both the column flow through and controlsamples were determined. Results were calculated as the OD₆₀₀ of theflow through divided by that of the control and expressed as apercentage of cells adsorbed to the column.

Example 10 Serum Bactericidal Assay

A serum bactericidal assay was performed as previously described (24)using pooled normal human serum at 10% final concentration (v/v) with 30min incubation at 37° C. Heat-inactivated normal human serum was used asa control.

Example 11 Immunoblots

Capsular polysaccharides of the serogroup A wild type and mynC mutantNmA001 were resolved on 15% DOC-PAGE gels and transferred onto PVDFmembrane using transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20%methanol). An identical gel was stained with Alcian blue to visualizecapsule. Membranes were blocked with 3% BSA in Tris-TWEEN (TWEEN 20 ispolyoxyethylene sorbitan monolaurate) buffer (0.5 M Tris, pH7.5, 0.9%NaCl, 0.05% TWEEN-20). Serogroup A capsule-specific monoclonal antibody14-1-A (13) was used as the primary antibody at a 1:1,000 dilution,while alkaline phosphatase conjugated goat anti-mouse IgG+IgM (OrganonTeknika Corporation, West Chester, Pa.) was used at 1:5,000 dilution.All incubations were done at room temperature for 1 hour. Blots weredeveloped in 20 ml of alkaline phosphatase buffer (0.1 M Tris, pH 9.5,0.1 M NaCl, 0.5 mM MgCl₂) containing 40 μl of 10% NBT in 70% DMF and 30μl of BCIP (50 mg/ml in DMF). Colony immunoblots were processedsimilarly using nitrocellulose membranes. After the meningococci werelifted, the membranes were allowed to air-dry for 30 min at roomtemperature and then blocked for 1 hour with 5% BSA in Tris-TWEENbuffer. Protein samples for western blots were resolved by 10% SDS-PAGEand transferred to PVDF membranes as described. Anti-penta-Hismonoclonal antibodies were used as primary antibodies at 1:1,000dilutions.

Example 12 Whole Cell ELISA

ELISAs were performed as described (11) with the followingmodifications: 50 μl aliquots of a 1:9 dilution of meningococcalsuspensions (OD₅₅₀=0.1) were applied to microtiter plates and driedovernight at 37° C. Monoclonal antibody 14-1-A was used at a 1:30,000dilution and alkaline phosphatase-conjugated goat-anti mouse secondaryantibody (Organon Teknika Corp. West Chester, Pa.) was used at a1:10,000 dilution. All incubations were performed at 37° C.

Example 13 Colorimetric Estimation of Capsule O-acetylation

O-acetylation of purified CPSs was measured colorimetrically asdescribed by Hestrin (25). Aliquots of CPS samples (500 μl) wereincubated with equal volume of 0.035 M hydroxylamine in 0.75 M NaOH for10 min at 25° C., and then 1 M of perchloric acid (500 μl) and 70 mMferric perchlorate in 0.5 M perchloric acid (500 μl) were added. Thepink color resulting from the presence of O-acetyl groups was quantifiedat 500 nm with a known amount of ethyl acetate as the standard.

Example 14 In Vitro O-acetyltransferase Activity

O-acetyltransferase enzyme activity was determined by autoradiographyusing ¹⁴C labeled acetyl co-enzyme A as acetyl donor and purifiedmeningococcal CPSs as substrate. In a typical 50 μl reaction volume, 50μg of CPS, 10 μg of the MynC protein and 0.5 μCi of [¹⁴C]-acetyl-CoA(0.05 μCi/μl, specific activity 47 μCi/μmol) were incubated in a buffercomposed of 10 mM Tris, pH 7.4, 20 mM NaCl, 1 mM MgCl₂, and 25 mM EDTA.The reaction mixtures were concentrated to near-dryness after 1 hourincubation at 37° C. and then re-suspended in 10 μl of water and 10 μlof 2× sample buffer. The samples were resolved with 15% DOC-PAGE gels.Gels were incubated with intensifying solution (Dupont) for 30 minbefore drying under vacuum. The dried gels were exposed to X-ray filmsat −80° C.

Example 15 Concentration, Time and pH Dependence

A typical 25 μl reaction containing 1 to 6 μg of purified MynC, 0.25 μCiof ¹⁴C acetyl CoA and 25 μg of OAc− CPS purified from mynC nonpolarmutant in the Tris MgCl₂ EDTA buffer noted above were incubated for 1 hat 37° C. After the reaction, the CPS was precipitated with 80% (v/vfinal concentration) ethanol, and the pellet was washed 3 times with 80%ethanol and air-dried. ¹⁴C acetyl incorporations were measured usingliquid scintillant (ScintiSafe Econo 1 Fisher Scientific) and a liquidscintillation analyzer (Packard Tricarb 2500 TR). The amount of ¹⁴Cacetyl incorporation into CPS by MynC was determined at 5, 15, 30, 60,120 and 180 min. At the respective time points, 100 μl of ethanol wasadded to the 25 μl reaction mixtures (see above) containing 5 μg ofpurified MynC protein, to precipitate the CPS. The pellets were washedthree times with 80% ethanol, air-dried, and the incorporation measuredby scintillation counts. The stability of 50 μg triplicate samples ofmutant CPS substrate was tested in the reaction condition without theenzyme along these time points by estimating the neutral sugar (20) inthe pellets after respective washes. In order to determine the optimalpH for the MynC activity, citrate buffer ranging 4.5 to 6.5, phosphatebuffer from pH 5.8 to 8.0 and borate buffer from 8.5 to 10.5 with finalsalt concentration of 20 mM were used in the 25 μl reaction (see above)noted above with 5 μg of purified MynC. The reaction was incubated for 1h at 37° C.

TABLE 1 Strains, plasmids, and primers used in this studyStrains/plasmids/ Reference/ Primers Description or sequence SourceN. meningitidis F8229 N. meningitidis serogroup A strain (CDC1750) (11)NmA001 NmA with chromosomal mynC::aphA-3 mutation NmAwtc1F8229 carrying pGS205 (mynC) NmAnpc1 NmA001 carrying pGS205 (mynC)E. coli DH5α Cloning strain (57) BLR21(DE3) pLysS Expression strainNovagen Plasmids pCR 2.1 TA cloning Stratagene pUC18Cloning vector, Amp^(r) (58) pUC18K Source of aphA-3 (km^(r)) cassette(59) pFlag-CTC Cloning vector for FLAG fusion Sigma pYT250Meningococcal shuttle vector (Em^(r)) (60) pGS201SE57-SE61 PCR product cloned into pCR2.1 pGS202aphA-3 cloned into blunted Sspl site of pGS201 pGS203Full length mynC obtained from SG005 (Ndel) and SG006(xhol) PCR product cloned into Ndel-Xhol digested pET20b pGS204Full length mynC with His-tag obtained from SG007 (HindIII)and SG008 (EcoRI) PCR product cloned into pCR2.1 pGS205HindIII-EcoRV digested fragment of pGS204 ligated withHindIII-Smal digested fragment of pFlag-CTC pGS206BglI digested fragment of pGS205 subcloned into EcoRV site of pYT250Primers             5′ → 3′ SE56 AATCATTTCAATATCTTCACAGCC; SEQ ID NO: 3SE57 TTACCTGAATTTGAGTTGAATGGC; SEQ ID NO: 4 SE61CAAAGGAAGTTACTGTTGTCTGC; SEQ ID NO: 5 YT79CATCATAACGGTTCTGGCAAATATTC; SEQ ID NO: 6 YT80CTGTATCAGGCTGAAAATCTTCTCTC; SEQ ID NO: 7 SG005GAACATATGTTATCTAATTTAAAAAAC; SEQ ID NO: 8 SG006TTACTCGAGATATATATTTTGGATTATGGT; SEQ ID NO: 9 SG007GGAGATATACATAAGCTTTCTAATTTAAAA; SEQ ID NO: 10 SG008AGCGAATTCTCAGTGGTGGTGGTGGTGGTG; SEQ ID NO: 11

TABLE 2 Homology of MynC (247aa) Similarity Organism Protein (aa)Function Identity (%) (%) Range Caldicellulosiruptor XynC, Acetyl Xylan27 45 208 saccharolyticus esterase degradation (266) Actinobacillus suisHypothetical Unknown 33 49 133 protein (410) Bacillus anthracisConserved Unknown 26 45 184 protein (896) Lactococcus lactis EpsK (152)EPS 30 44 130 biosynthesis Staphylococcus Cap8I (464) CPS 25 46 119aureus biosynthesis

TABLE 3 Proton assignments in ppm of the 3-O—Ac and non-O—Ac CPSs. CPSCH₃—NAc CH₃—OAc H-1 H-2 H-3 H-4 H-5 H6/6′ 3-O—Ac 2.08 2.06/2.10 5.464.61 5.20 4.01 4.14 4.20/4.30 Non OAc 2.08 — 5.44 4.45 4.14 3.82 4.014.18/4.24

TABLE 4 Relative percentages* of the various CPSs from wild type, mynC3-O—Ac 4-O—Ac Strain CPS^(a) CPS^(b) 4-OAc CPS^(c) Non-OAc CPS Wild type40 10 17 33 mynC::aphA3 0 0 0 100 NmAnpc1 26 4.8 8.4 61 *Calculated fromthe integration values of the H2 resonances. ^(a)O—Ac, O-acetylated.^(b)Based on the assignment of the resonance of the H2 of 4-O—Ac-ManNAcwhen it is adjacent to a 3-O—Ac-ManNAc residue. ^(c)Based on theassignment of the resonance of the H2 of 4-O—Ac-ManNAc when it isadjacent to a non-O—Ac-ManNAc residue.

REFERENCES CITED IN THE SPECIFICATION

-   1. Liu, T. Y., Gotschlich, E. C., Jonssen, E. K., and    Wysocki, J. R. (1971) J. Biol. Chem. 246, 2849-2858.-   2. Bundle, D. R., Smith. I. C. P., and Jennings. H. J. (1974) J.    Biol. Chem. 249, 2275-2281.-   3. Bhattacharjee, A. K., Jennings, H. J., Kenny, C. P., Martin, A.,    and Smith, I. C. (1976) Can. J. Biochem. 54, 1-8.-   4. Bhattacharjee, A. K., Jennings, H. J., Kenny, C. P., Martin, A.,    and Smith, I. C. P. (1975) J. Biol. Chem. 250, 1926-1932.-   5. Claus, H., Borrow, R., Achtman, M., Morelli, G., Kantelberg, C.,    Longworth, E., Frosch, M., and Vogel, U. (2004) Mol. Microbiol. 51,    227-239.-   6. Orskov, F., Orskov, I., Sutton, A., Schneerson, R., Lin, W.,    Egan, W., Hoff, G. E., and Robbins, J. B. (1979) J. Exp. Med. 149,    669-685.-   7. Szu, S. C., Li, X. R., Stone, A. L., and Robbins, J. B. (1991)    Infect. Immun. 59, 4555-4561.-   8. Berry, D. S., Lynn, F., Lee, C. H., Frasch, C. E., and    Bash, M. C. (2002) Infect. Immun. 70, 3707-3713.-   9. Roberts, I. S. (1996) Annu. Rev. Microbiol. 50, 285-315.-   10. Whitfield, C., and Roberts, I. S. (1999) Mol. Microbiol. 31,    1307-1319.-   11. Swartley, J. S., Liu, L. J., Miller, Y. K., Martin, L. E.,    Edupuganti, S., and Stephens, D. S. (1998) J. Bacteriol. 180,    1533-1539.-   12. Pinner, R. W., Onyango, F., Perkins, B. A., Mirza, N. B.,    Ngacha, D. M., Reeves, M., DeWitt, W., Njeru, E., Agata, N. N., and    Broome, C. V. (1992) J. Infect. Diseases 166, 359-364.-   13. Zollinger, W. D., Boslego, J., Froholm, L. O., Ray, J. S.,    Moran, E. E., and Brandt, B. L. (1987) Antonie Van Leeuwenhoek 53,    403-411.-   14. Janik, A., Juni, E., and Heym, G. A. (1976) J. Clin. Microbiol.    4, 71-81.-   15. Dorsey, C. W., Tolmasky, M. E., Crosa, J. H., and    Actis, L. A. (2003) Microbiology 149, 1227-1238.-   16. Clark, V. L., Campbell, L. A., Palermo, D. A., Evans, T. M., and    Klimpel, K. W. (1987) Infect. & Immun. 55, 1359-1364.-   17. de Maagd, R. A., and Lugtenberg, B. (1986) J. Bacteriol 167,    1083-1085.-   18. Finberg, K. E., Muth, T. R., Young, S. P., Maken, J. B.,    Heitritter, S. M., Binns, A. N., and Banta, L. M. (1995) J.    Bacteriol. 177, 4881-4889.-   19. Gotschlich, E. C., Liu, T. Y., and Artenstein, M. S. (1969) J.    Exp. Med. 129, 1349-1365.-   20. Dubois, M. (1956) Anal. Chem. 28, 350-356.-   21. Reuhs, B. L., Carlson, R. W., and Kim, J. S. (1993) J.    Bacteriol. 175, 3570-3580.-   22. Stevenson, T. T., and Furneaux, R. H. (1991) Carbohydr. Res. 11,    195-211.-   23. Karlyshev, A. V., Linton, D., Gregson, N. A., Lastovica, A. J.,    and Wren, B. W. (2000) Mol. Microbiol. 35, 529-541.-   24. Kahler, C. M., Martin, L. E., Shih, G. C., Rahman, M. M.,    Carlson, R. W., and Stephens, D. S. (1998) Infect. Immun. 66,    5939-5947.-   25. Hestrin, S. (1949) J. Biol. Chem. 180, 249-261.-   26. Luthi, E., Love, D. R., McAnulty, J., Wallace, C., Caughey, P.    A., Saul, D., and Bergquist, P. L. (1990) Appl. Environ. Microbiol.    56, 1017-1024.-   27. Sau, S., Sun, J., and Lee, C. Y. (1997) J. Bacteriol. 179,    1614-1621.-   28. Lernercinier, X., and Jones, C. (1996) Carbohydr. Res. 296,    83-96.-   29. Jones, C., and Lernercinier, X. (2002) J. Pharm. Biomed. Anal.    30, 1233-1247.-   30. Richmond, P., Borrow, R., Findlow, J., Martin, S., Thornton, C.,    Cartwright, K., and Miller, E. (2001) Infect. Immun. 69, 2378-2382.-   31. Longworth, E., Fernsten, P., Mininni, T. L., Vogel, U., Claus,    H., Gray, S., Kaczmarski, E., and Borrow, R. (2002) FEMS. Immunol.    Med. Microbiol. 32, 119-123.-   32. Richmond, P., Goldblatt, D., Fusco, P. C., Fusco, J. D., Heron,    I., Clark, S., Borrow, R., and Michon, F. (1999) Vaccine 18,    641-646.-   33. McNeely, T. B., Staub, J. M., Rusk, C. M., Blum, M. J., and    Donnelly, J. J. (1998) Infect. Immun. 66, 3705-3710.-   34. Franklin, M. J., and Ohman, D. E. (2002) J. Bacteriol. 184,    3000-3007.-   35. Nivens, D. E., Ohman, D. E., Williams, J., and    Franklin, M. J. (2001) J. Bacteriol. 183, 1047-1057.-   36. Pier, G. B., Coleman, F., Grout, M., Franklin, M., and    Ohman, D. E. (2001) Infect. Immun. 69, 1895-1901.-   37. Bloemberg, G. V., J. E. Thomas-Oates, B. J. J. Lugtenberg,    and H. P. Spaink. (1994) Mol. Microbiol. 11, 793-804.-   38. Lopez-Lara, I. M., van den Berg, J. D., Thomas-Oates, J. E.,    Glushka, J., Lugtenberg, B. J., and Spaink, H. P. (1995) Mol.    Microbiol. 15, 627-638.-   39. Spaink, H. P., Sheeley, D. M., van Brussel, A. A., Glushka, J.,    York, W. S., Tak, T., Geiger, O., Kennedy, E. P., Reinhold, V. N.,    and Lugtenberg, B. J. (1991) Nature 354, 125-130.-   40. Antignac, A., Ducos-Galand, M., Guiyoule, A., Pires, R.,    Alonso, J. M., and Taha, M. K. (2003) Clin. Infect. Dis. 37,    912-920.-   41. Girardin, S. E., Travassos, L. H., Herve, M., Blanot, D.,    Boneca, I. G., Philpott, D. J., Sansonetti, P. J., and    Mengin-Lecreulx, D. (2003) J Biol Chem. 278, 41702-41708.-   42. Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F.,    Crespo, J., Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M.,    Foster, S. J., Moran, A. P., Fernandez-Luna, J. L., and    Nunez, G. (2003) J. Biol. Chem. 278, 5509-5512.-   43. Hindson, V. J., Moody, P. C., Rowe, A. J., and    Shaw, W. V. (2000) J. Biol. Chem. 275, 461-466.-   44. Hindson, V. J., Dunn, S. O., Rowe, A. J., and Shaw, W. V. (2000)    Biochim. Biophys. Acta 1479, 203-213.-   45. Lewendon, A., Ellis, J., and Shaw, W. V. (1995) J. Biol. Chem.    270, 26326-26331.-   46. Denk, D., and Bock, A. (1987) J. Gen. Microbiol. 133 (Pt 3),    515-525.-   47. Wigley, D. B., Derrick, J. P., and Shaw, W. V. (1990) FEBS Lett.    277, 267-271.-   48. Hara, O., and Hutchinson, C. R. (1992) J. Bacteriol. 174,    5141-5144.-   49. Luck, P. C., Freier, T., Steudel, C., Knirel, Y. A., Luneberg,    E., Zahringer, U., and Helbig, J. H. (2001) Int. J. Med. Microbiol.    291, 345-352.-   50. Slauch, J. M., Lee, A. A., Mahan, M. J., and    Mekalanos, J. J. (1996) J. Bacteriol. 178, 5904-5909.-   51. Verma, N. K., Brandt, J. M., Verma, D. J., and    Lindberg, A. A. (1991) Mol. Microbiol. 5, 71-75.-   52. Firmin, J. L., Wilson, K. E., Carlson, R. W., Davies, A. E., and    Downie, J. A. (1993) Mol. Microbiol. 10, 351-360.-   53. Bhasin, N., Albus, A., Michon, F., Livolsi, P. J., Park, J. S.,    and Lee, J. C. (1998) Mol. Microbiol. 27, 9-21.-   54. Higa, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8872-8878.-   55. Kroon, P. A., Williamson, G., Fish, N. M., Archer, D. B., and    Belshaw, N. J. (2000) Eur. J. Biochem. 267, 6740-6752.-   56. Yi, K., Stephens, D. S., and Stojiljkovic, I. (2003) Infect.    Immun. 71, 1849-1855.-   57. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580.-   58. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33,    103-119.-   59. Menard, R., Sansonetti, P. J., and Parsot, C. (1993) J.    Bacteriol. 175, 5899-5906.-   60. Tzeng, Y. L., Datta, A., Kolli, V. K., Carlson, R. W., and    Stephens, D. S. (2002) J. Bacteriol. 184, 2379-2388.-   61. Jennings, H. J., A. K. Bhattacharjee, D. R. Bundle, C. P.    Kenny, A. Martin, and I. C. Smith. 1977. Journal of Infectious    Diseases 136 Suppl:S78-83.-   62. Stephens, D. S., L. H. Hoffman, and Z. A. McGee. 1983. J.    Infect. Dis. 148:369-76.-   63. Drogari-Apiranthitou, M., E. J. Kuijper, N. Dekker, and J.    Dankert. 2002. Infect Immun 70:3752-8.-   64. Filice, G. A., P. S. Hayes, G. W. Counts, J. M. Griffiss,    and D. W. Fraser. 1985. J Clin Microbiol 22:152-6.-   65. Amir, J., L. Louie, and D. M. Granoff. 2005. Vaccine 23:977-83.-   66. Fattom, A. I., J. Sarwar, L. Basham, S. Ennifar, and R.    Naso. 1998. Infect Immun 66:4588-92.-   67. Orskov, F., I. Orskov, A. Sutton, R. Schneerson, W. Lin, W.    Egan, G. E. Hoff, and J. B. Robbins. 1979. J Exp Med 149:669-85.-   68. Szymanski C. M., Michael F. S., Jarrell H. C., et al. 2003. J.    Biol. Chem. 278: 24509-20.-   69. Gudlavalleti S. K., Datta A. K., Tzeng Y. L., Noble C.,    Carlson R. W., Stephens D. S. 2004. J. Biol. Chem. 279:42765-73.

1. A method for acetylating Serogroup A polysaccharide prepared fromNeisseria meningitidis, said method comprising the step of contacting anisolated serogroup A meningococcal capsular polysaccharide with anisolated Serogroup A Neisseria meningitidis O-acetyltransferasepolypeptide comprising SEQ ID NO:2.
 2. The method of claim 1 wherein theO-acetyltransferase polypeptide is encoded by an isolated polynucleotidesequence comprising SEQ ID NO:1.
 3. An improved immunogenic compositioncomprising an acetylated capsular polysaccharide of Neisseriameningitidis, wherein the improvement comprises acetylation of thecapsular polysaccharide according to the method of claim
 1. 4. Theimmunogenic composition of claim 3 further comprising an adjuvant orcytokine.
 5. A method for the O-acetylation of a meningococcal serogroupA capsular polysaccharide, the method comprising contacting apreparation comprising the meningococcal serogroup A capsularpolysaccharide with an isolated Serogroup A Neisseria meningitidisO-acetyltransferase polypeptide comprising SEQ ID NO:2.
 6. The method ofclaim 5, wherein the O-acetyltransferase polypeptide is encoded by anisolated polynucleotide sequence comprising SEQ ID NO:1.
 7. An isolatedacetylated meningococcal serogroup A capsular polysaccharide, whereinthe isolated meningococcal serogroup A capsular polysaccharide has beenacetylated according to the method of claim
 5. 8. The isolatedacetylated meningococcal serogroup A capsular polysaccharide of claim 7,wherein the acetylated meningococcal serogroup A capsular polysaccharideis 90-95% acetylated.
 9. A composition comprising the isolatedacetylated meningococcal serogroup A capsular polysaccharide of claim 7.10. The composition of claim 9 further comprising an adjuvant orcytokine.
 11. An acetylated capsular polysaccharide of Neisseriameningitidis serogroup A, wherein the O-3 and/or O-4 acetylation of theacetylated capsular polysaccharide of Neisseria meningitidis serogroup Ais increased over wild type Neisseria meningitidis serogroup A whereinthe acetylation is performed by an O-acetyltransferase polypeptidecomprising the sequence of SEQ ID NO:2.
 12. An immunogenic compositioncomprising the acetylated capsular polysaccharide of Neisseriameningitidis serogroup A of claim
 11. 13. The immunogenic composition ofclaim 12 further comprising an immunogenic carrier.
 14. The immunogeniccomposition of claim 12 further comprising an adjuvant or cytokine. 15.A method for acetylating the O-3 and/or O-4 positions of an (α1→6)linked N-acetyl-D-mannosamine-1-phosphate polymer, said methodcomprising the step of contacting an (α1→6) linkedN-acetyl-D-mannosamine-1-phosphate polymer with an O-acetyltransferasepolypeptide comprising SEQ ID NO:2.
 16. The method of claim 15 whereinthe O-acetyltransferase polypeptide is encoded by an isolatedpolynucleotide sequence comprising SEQ ID NO:1.
 17. An isolatedacetylated (α1→6) linked N-acetyl-D-mannosamine-1-phosphate polymer,wherein the isolated (α1→6) linked N-acetyl-D-mannosamine-1-phosphatepolymer has been acetylated according to the method of claim
 15. 18. Theisolated acetylated (α1→46) linked N-acetyl-D-mannosamine-1 phosphatepolymer of claim 17, wherein the acetylated (α1→6) linkedN-acetyl-D-mannosamine-1 phosphate polymer is 90-95% acetylated.
 19. Acomposition comprising the isolated acetylated an (α1→6) linkedN-acetyl-D-mannosamine-1-phosphate polymer of claim
 17. 20. Thecomposition of claim 19 further comprising an adjuvant or cytokine.